Materials and methods to potentiate cancer treatment

ABSTRACT

Compounds that inhibit DNA-dependent protein kinase, compositions comprising the compounds, methods to inhibit the DNA-PK biological activity, methods to sensitize cells the agents that cause DNA lesions, and methods to potentiate cancer treatment are disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of provisional U.S. PatentApplication No. 60/229,899, filed Sep. 1, 2000.

FIELD OF THE INVENTION

[0002] The present invention relates to inhibitors of DNA-dependentprotein kinase (DNA-PK), and to methods of using the inhibitors topotentiate cancer treatment.

BACKGROUND OF THE INVENTION

[0003] All cells possess mechanisms to maintain integrity of thecellular genome through detection and repair of, for example, adductformation, cross-linking, single-strand breaks, and double-strandbreaks. The mechanisms of detection and damage repair, collectively, arecalled DNA repair. DNA repair functions are carried out on lesions thatarise from exposure to a variety of environmental chemical and physicalagents, as well as from toxic agents generated intracellularly in normalcellular metabolism. Because DNA provides the information required forcell, tissue, and organism function, a large amount of cellular energyis devoted to maintaining intact structure of the genome.

[0004] The most genotoxic damages are those which induce DNA chaindisruptions, particularly double-strand breaks. DNA double-strand breaks(dsbs) can be induced by chemical or physical agents, includingintercalating agents, electrophilic compounds and ionizing radiation. Atleast two pathways responsible for the repair of DNA dsbs exist, i.e.,homologous recombination (HR) and nonhomologous end joining (NHEJ). Theformer reaction requires undamaged DNA from the homologous chromosome tobe used as a template in the repair of the DNA discontinuity. NHEJ, incontrast, is DNA homology independent and simply requires two free DNAends to be re-ligated. The exact molecular mechanisms by which both HRand NHEJ are effected remain to be elucidated.

[0005] DNA dsbs also are generated during the course of normal cellulardevelopment in some tissues. This observation first was appreciatedfollowing the discovery and characterization of the severe combinedimmuno-deficiency (scid) mouse. The scid syndrome is a genetic disorderwhich manifests as an absence of B- and T-cell immunity (Bosma et al.,Nature, 301:527-530 (1983), and reviewed in Bosma and Carroll, Annu.Rev. Immunol., 9:323-350 (1991)). The scid mouse is defective in theearliest stages of lymphoid cell development as a result of an inabilityto correctly rearrange T-cell receptor (TCR) and IgM μ chain DNA (Bosmaand Carroll, Annu. Rev. Immunol., 9:323-350 (1991), Dorshkind et al., J.Immunol, 132:1804-1808 (1984), Lauzon et al., J. Exp. Med.,164:1797-1802 (1986), Schuler et al., Cell, 46:963-972 (1986), Tutt etal., J. Immunol., 138:2338-2344 (1987), Lieber et al., Cell, 55:7-16(1988)). As a result, T- and B-cells do not progress beyond the CD25⁺CD4⁻ CD8⁻ and CD25⁻ pro-B cell stages, respectively. Site-specific V(D)Jrecombination is initiated in scid mice through the activity of the RAG1and RAG2 gene products, however, resolution of recombinationintermediates is disrupted (Fulop and Phillips, Nature, 347:479-482(1990), Biedermann et al., Proc. Natl. Acad. Sci. USA, 88:1394-1397(1991), Hendrickson et al., Proc. Natl. Acad. Sci. USA, 88:4061-4065(1991), Oettinger et al., Science, 248:1517-1522 (1990), Mombaerts etal., Cell, 68:869-877 (1992), Shinkai et al., Cell, 68:855-867 (1992),van Gent et al., Cell, 81:925-934 (1995), and reviewed in Lieber, FASEBJ., 5:2934-2944 (1991)). Nonproductive rearrangements in scid cellstypically result in large deletions at the TCR and Ig loci, while theprocessing of recombination signal sequences is not affected in thesecells. The scid mutation, therefore, specifically disrupts the formationof recombinant coding junctions (Lieber et al., Cell, 55:7-16 (1988),Malynn et al., Cell, 54:453-460 (1988)).

[0006] The defect in the scid mouse is caused by mutation of the geneencoding the catalytic subunit of the DNA-dependent protein kinase(DNA-PK) (Blunt et al., Cell, 80:813-823 (1995), Peterson et al., Proc.Nati. Acad. Sci. USA, 92:3171-3174 (1995)). Specifically, a nonsensemutation at tyrosine-4046 results in the deletion of the last 83 aminoacid residues (Blunt et al., Proc. Natl. Acad. Sci. USA, 93:10285-10290(1996), Danska et al., Mol. Cell. Biol., 16:5507-5517 (1996), Araki etal., Proc. Natl. Acad. Sci. USA, 94:2438-2443 (1997)).

[0007] DNA-PK is a trimeric complex composed of a p460 catalytic subunitand Ku80 (86 kDa) and Ku70 regulatory proteins. Ku70 and Ku80 wereinitially described as human autoantigens and function as cofactors invitro stimulating protein kinase activity through binding DNA (Mimori,J. Clin. Invest., 68:611-620 (1981), Dvir et al., Proc. Natl. Acad. Sci.USA, 89:11920-11924 (1992), Gottlieb and Jackson, Cell, 72:131-142(1993)). Ku70 and Ku80 exhibit highest affinity for DNA duplex terminiand gaps (Blier et al., J. Biol. Chem., 268:7594-7601 (1993), Falzon etal., J. Biol. Chem., 268: 10546-10552 (1993)). Although, the precisefunction of DNA-PK and its natural substrates remain unknown, thisenzyme phosphorylates a number of proteins in vitro, including manytranscription factors and p53 (Lees-Miller et al., Mol. Cell. Biol.,12:5041-5049 (1992), Anderson and Lees-Miller, Crit. Rev. Euk. GeneExp., 2:283-314 (1992)).

[0008] Cultured scid cells are sensitive to killing by agents thatinduce DNA double-strand breaks (dsbs), indicating a role for DNA-PK inthe repair of these lesions. The scid defect also sensitizes mice toradiation-induced lymphomagenesis (Lieberman et al., J. Exp. Med.,176:399-405 (1992)). Lymphomas arise in scid mice at frequencies rangingfrom 50 to 100% at x-ray doses that do not affect wild-type mice. Sinceunirradiated scid mice are not particularly sensitive tolymphomagenesis, the background level of tumor-inducing dsbs must eitherbe low enough to be effectively repaired or the damaged cells areeffectively eliminated.

[0009] The therapeutic benefit of radiation and chemotherapy in thetreatment of cancer is well documented. These physical and chemicalagents act by disrupting DNA metabolism at the level of DNA structure,synthesis, transcription and chromosome transmission. Most of theseagents act by inducing DNA-specific lesions. Presumably, if tumor cellsare sensitive to therapies that introduce DNA specific lesions, thenthese therapies will be made more effective by simultaneously disruptingthe cellular repair of these damages. Therefore, inhibition of cellularDNA-PK activity following treatment with agents that induces DNA dsbswill potentiate the therapeutic index of these agents.

[0010] Thus, there exists a need in the art to identify compounds thatcan improve the efficiency of radiation and chemotherapy in treatment ofcancer. Identification of DNA-PK inhibitors can permit development oftreatment regimens that include lower doses of radiation and/orchemotherapy drugs, thereby reducing the unwanted side effects thatoften accompany the treatments.

SUMMARY OF THE INVENTION

[0011] The invention provides compounds having a DNA-PK inhibitingactivity. The present DNA-PK inhibitors can be used in diagnostic andtherapeutic methods useful in the field of cancer therapy. Moreparticularly, the DNA-PK inhibitors permit development of compositionsand treatment regimens that can be used with doses of radiation and/orchemotherapy drugs lower than a standard prescribed dose. The reducedexposure to radiation and chemotherapy drugs improves a patient'sprognosis with regard to unwanted adverse side effects that oftenaccompany cancer treatments.

[0012] The DNA-PK inhibitors of the invention are compounds having aformula (I):

[0013] or a pharmaceutically acceptable salt thereof, wherein:

[0014] n is an integer 0 through 4;

[0015] Z, independently, is CR³, or N;

[0016] A is an optionally substituted four- to seven-membered aliphaticring containing 0, 1, 2, or 3 heteroatoms, independently selected fromthe group consisting of N, O, and S;

[0017] R¹ is selected from the group consisting of hydrogen, alkyl,substituted alkyl, cycloalkyl, heterocycloalkyl, N(R^(h))₂, OR^(h),carboxyl, carboxy, nitro, hydrazono, hydroxyamino, cyano, aldehyde,carboxarmide, thiocarboxamide, acyl, mercapto, sulfonyl,trifluoromethyl, heteroaryl, and substituted heteroaryl;

[0018] R² is selected from the group consisting of hydrogen, alkyl,substituted alkyl, carbamoyl, carboxamide, N(R^(h))₂, carboxy, OR^(h),sulfamyl, nitro, phosphate, and sulfonamido; or

[0019] R¹ and R² are taken together with the carbon atoms to which eachis attached to form a 5-, 6-, or 7-membered ring, wherein 1, 2, or 3carbon atoms of R¹ and R² optionally are a heteroatom selected from thegroup consisting of O, N, S, and P, said ring optionally substitutedwith one or more ═O, ═S, ═NH, OR^(b), N(R^(h))₂, carboxyl, carboxy,alkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl,said heteroatom optionally substituted with a group selected from thegroup consisting of aryl, substituted aryl, alkyl, alkyl substitutedwith acyl, and acyl;

[0020] R³, independently, is selected from the group consisting ofhydrogen, halo, aldeyhde, OR^(h), nitro, N(R^(h))₂, carboxyl, carboxy,sulfonamido, sufamyl, and sulfo or a halide derivative thereof,

[0021] wherein R^(h), independently, is selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, aryl,substituted aryl, heteroaryl, and substituted heteroaryl; and

[0022] R⁴, independently, is selected from the group consisting ofOR^(h), halo, N(R^(h))₂, aldehyde, alkyl, substituted alkyl, acyl, aryl,substituted aryl, heteroaryl, and substituted heteroaryl;

[0023] with the proviso that when A is morpholinyl, R² and R⁴ arehydrogen, and ZR³ is CH at each occurrence, then R¹ is different from—(CO)—CH₃, (C═CH₂)-phenyl, and nitro; and with the proviso that when Ais morpholinyl, R⁴ is hydrogen, and Z is nitrogen at each occurrence,then R¹ and R², when taken together, is different from triazole.

[0024] Additional compounds useful as DNA-PK inhibitors have astructural formula (II):

[0025] or a pharmaceutically acceptable salt thereof, wherein:

[0026] Z, independently, is CR⁷ or N;

[0027] L is selected from the group consisting of alkylene, substitutedalkylene, carbonyl, carbamoyl, NR^(h), oxy (—O—), thio (—S—), thionyl(—SO—), and sulfonyl;

[0028] A is absent, or A is an optionally substituted four- toseven-membered aliphatic ring containing 0, 1, 2, or 3 heteroatoms,independently selected from the group consisting of N, O, and S;

[0029] R⁵ is selected from the group consisting of hydrogen, alkyl,substituted alkyl, cycloalkyl, heterocycloalkyl, N(R^(h))₂, OR^(h),carboxyl, carboxy, nitro, hydrazono, hydroxyamino, cyano, aldehyde,carboxamide, thiocarboxamide, acyl, mercapto, sulfonyl, trifluoromethyl,heteroaryl, and substituted heteroaryl;

[0030] R⁶ is selected from the group consisting of hydrogen, alkyl,substituted alkyl, carbamoyl, carboxamide, N(R^(h))₂, carboxy, OR^(h),sulfamyl, nitro, phosphate, and sulfonamido; or

[0031] R⁵ and R⁶ are taken together with the carbon atoms to which eachis attached to form a 5-, 6-, or 7-membered ring, wherein 1, 2, or 3carbon atoms of R⁵ and R⁶ optionally are a heteroatom selected from thegroup consisting of O, N, S, and P, said ring optionally substitutedwith one or more of ═O, ═S, ═NH, OR^(h), N(R^(h))₂, carboxyl, carboxy,alkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl,and said heteroatom optionally substituted with a substituent selectedfrom the group consisting of aryl, substituted aryl, alkyl, alkylsubstituted with acyl, and acyl;

[0032] R⁷, independently, is selected from the group consisting ofhydrogen, halo, aldehyde, OR^(h), nitro, N(R^(h))₂, carboxyl, carboxy,sulfamyl, sulfonamido, and sulfo or a halide derivative thereof,

[0033] wherein R^(h), independently, is selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, aryl,substituted aryl, heteroaryl, and substituted heteroaryl; and

[0034] R⁸, independently, is selected from the group consisting ofOR^(h), halo, N(R^(h))₂, aldehyde, alkyl, substituted alkyl, acyl, aryl,substituted aryl, heteroaryl, and substituted heteroaryl.

[0035] The invention further provides a pharmaceutical compositioncomprising (a) one or more DNA-PK inhibitors of formula (I) or (II) and(b) a pharmaceutically acceptable carrier. The pharmaceuticalcomposition optionally comprises an anti-neoplastic agent.

[0036] The invention also provides pharmaceutical compositionscomprising (a) one or more DNA-PK inhibitors of formula (I) or (II) and(b) a radiotherapeutic (or anti-neoplastic agent). Radiotherapeuticagents include compounds that can be targeted to neoplastic cell typesand include one or more attached radioisotopes.

[0037] The invention also provides methods of inhibiting DNA-PKactivity. The method comprises the step of contacting a DNA-PK with oneor more compounds of formula (I) or (II).

[0038] The invention further provides methods of sensitizing a cell toan agent that induces a DNA lesion comprising the step of contacting thecell with one or more DNA-PK inhibitors of formula (I) or (II). In oneaspect, the agent that induces a DNA lesion is selected from the groupconsisting of radiation, exogenous chemicals, metabolite by-products,and combinations thereof

[0039] The invention further provides methods of potentiating atherapeutic regimen for treatment of cancer comprising the step ofadministering to an individual in need thereof an effective amount of aDNA-PK inhibitor of formula (I) or (II). In one aspect, methods includethose wherein the therapeutic regimen for treatment of cancer isselected from the group consisting of chemotherapy, radiation therapy,and a combination of chemotherapy and radiation therapy. In methodswherein the therapeutic regimen includes chemotherapy, the DNA-PKinhibitor is administered before, concurrently with, and/or afteradministration of the chemotherapeutic agent. The therapeutic regimenalso further can include any other conventional or experimental therapy,including for example, nutritional and/or surgical techniques.

[0040] The invention also provides methods of characterizing the potencyof a test compound as an inhibitor of a DNA-PK polypeptide, said methodcomprising the steps of: (a) measuring activity of a DNA-PK polypeptidein the presence of a test compound; (b) comparing the activity of theDNA-PK polypeptide in the presence of the test compound to the activityof the DNA-PK enzyme in the presence of an equivalent amount of areference compound of formula (I) or (II), wherein a lower activity ofthe DNA-PK polypeptide in the presence of the test compound than in thepresence of the reference compound indicates that the test compound is amore potent inhibitor than the reference compound, and a higher activityof the DNA-PK polypeptide in the presence of the test compound than inthe presence of the reference compound indicates that the test compoundis a less potent inhibitor than the reference compound.

[0041] The invention further provides methods of characterizing thepotency of a test compound as an inhibitor of a DNA-PK polypeptide, saidmethod comprising the steps of: (a) determining an amount of a controlcompound of formula (I) or (II) that inhibits an activity of a DNA-PKpolypeptide by a reference percentage of inhibition, thereby defining areference inhibitory amount for the control compound; (b) determining anamount of a test compound that inhibits an activity of a DNA-PKpolypeptide by a reference percentage of inhibition, thereby defining areference inhibitory amount for the test compound; (c) comparing thereference inhibitory amount for the test compound to a referenceinhibitory amount determined according to step (a) for the controlcompound of formula (I) or (II), wherein a lower reference inhibitoryamount for the test compound than for the control compound indicatesthat the test compound is a more potent inhibitor than the controlcompound, and a higher reference inhibitory amount for the test compoundthan for the control compound indicates that the test compound is a lesspotent inhibitor than the control compound. The method utilizes areference inhibitory amount, which is the amount of the compound thatinhibits the activity of the DNA-PK polypeptide by 50%, by 60%, by 70%,or by 80%. In another aspect, the method employs a reference inhibitoryamount that is the amount of the compound that inhibits the activity ofthe DNA-PK polypeptide by 90%, by 95%, or by 99%. Methods of theinvention can comprise determining the reference inhibitory amount ofthe test compound in an in vitro biochemical assay, determining thereference inhibitory amount of the test compound in an in vitrocell-based assay, or determining the reference inhibitory amount of thetest compound in an in vivo assay.

[0042] The invention also provides an article of manufacture comprising:(a) an anti-cancer compound that induces double-strand DNA breakage incells, and (b) a package insert describing coordinated administration toa patient of said anti-cancer compound and a DNA-PK inhibitor compoundof formula (I) or (II). The article of manufacture comprises ananti-cancer compound, preferably a chemotherapeutic compound, preferablyselected from the group consisting of bleomycin, etoposide, andchlorambucil.

[0043] The invention further provides an article of manufacture,comprising: (a) a compound selected from the group consisting ofcytokines, lymphokines, growth factors, and hematopoietic factors; and(b) a package insert describing coordinated administration to a patientof said compound and a DNA-PK inhibitor compound selected from compoundsof formula (I) or (II).

DETAILED DESCRIPTION OF THE INVENTION

[0044] Definitions

[0045] An “IC₅₀ value” of a compound is defined as the concentration ofthe compound required to produce 50% inhibition of DNA-PK biological orenzymatic activity. Inhibitors of DNA-PK activity are defined to have anIC₅₀ of preferably less than about 200 μM, less than about 100 μM, lessthan about 50 μM, and from about 0.005 μM to 40 μM. Most preferably, apresent inhibitor has an lC₅₀ of less than 1 μM.

[0046] The term “pharmaceutically acceptable carrier” as used hereinrefers to compounds suitable for use in contact with recipient animals,preferably mammals, and more preferably humans, and having a toxicity,irritation, or allergic response commensurate with a reasonablebenefit/risk ratio, and effective for their intended use.

[0047] The term “prodrug” as used herein refers to compounds whichtransformed rapidly in vivo to a compound of the invention, for example,by hydrolysis. Prodrugs of the invention also can be active in theprodrug form. A thorough discussion is provided in Higuchi et al.,Prodrugs as Novel Delivery Systems, Vol. 14, of the A.C.S.D. SymposiumSeries, and in Roche (ed), Bioreversible Carriers in Drug Design,American Pharmaceutical Association and Pergamon Press, 1987.

[0048] The term “alkyl” and “alkylene” as used herein refers to astraight- or branched-chain hydrocarbon group, preferably containing oneto eight carbon atoms. Examples of suitable alkyl groups are C₁-C₅ alkylgroups. As used herein the designation C_(x)-C_(y) and C_(x-y), whereinx and y are integers, denotes an alkyl group having from x to y carbons,e.g., a C₁-C₅ or C₁₋₅ alkyl group has one to five carbon atoms.Particular examples of alkyl groups include, but are not limited to,methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl (1-methylpropyl),t-butyl(1,1 -dimethylethyl), n-pentyl, t-pentyl(1,1 -dimethylpropyl),n-hexyl, and the like. Examples of alkylene groups include methylene(—CH₂—) and ethylene (—CH₂CH₂—).

[0049] The term “substituted alkyl” and “substituted alkylene” as usedherein refers to an alkyl or alkylene group having one or moresubstituents. The substituents can include, but are not limited to,cycloalkyl, aryl, heteroaryl, heterocyclic, substituted aryl,substituted heteroaryl, substituted heterocyclic, N(R^(h))₂, OR^(h),SR^(h), sulfoxide, sulfonyl, halo, carboxyl, acyl, carboxy, hydrazino,hydrazono, and hydroxyamino. Like alkyl groups, the preferredsubstituted alkyl groups have one to five carbons, not including carbonatoms on the substituent group. Preferably, the alkyl group is eithermono- or di- substituted at one, two, or three carbons. The substituentscan be bound to the same carbon or different carbons.

[0050] The term “alkoxy” as used herein refers to a straight- orbranched-chain alkyl or substituted alkyl group attached to the parentmolecule through an oxygen atom, typically by a carbon to oxygen bond.The hydrocarbon of the alkoxy group preferably contains one to fivecarbon atoms. Typical alkoxy groups are methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, sec-butoxy (1-methylpropoxy),t-butoxy(1,1-dimethylethoxy), n-pentoxy, t-pentoxy(1,1-dimethylpropoxy),and the like. The term “thioalkoxy” is similarly defined, except sulfurreplaces oxygen.

[0051] The term “acyl” as used herein refers to an R^(a)C(═O)-groupattached to the parent molecule through a carbonyl (—C═O) group. R^(a)is defined as an alkyl, substituted alkyl, cycloalkyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocylic, and substitutedheterocyclic group. The preferred acyl group contains one to ten carbonatoms.

[0052] The term “cycloalkyl” as used herein refers to nonaromatic cyclichydrocarbon group, preferably containing three to six carbon atoms.Examples of cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, and the like. Cycloalkyl groups also can have alkyl andalkoxy substituents, as defined above, as well as halo substituents.

[0053] The term “aldehyde” as used herein refers to a —CHO group.

[0054] The term “amino” as used herein refers an —NH₂ or —NH— group,wherein each hydrogen in each formula can be replaced with an alkyl,aryl, heteroaryl, heterocyclic, substituted alkyl, substituted aryl, orsubstituted heterocyclic group, i.e., N(R^(h))₂. In the case of —NH₂,the hydrogen atoms also can be replaced with a substituents takentogether to form a 5- to 6-membered aromatic or nonaromatic ring,wherein one or two carbons of the ring optionally are replaced with aheteroatom selected from the group consisting of sulfur, oxygen, andnitrogen. The ring also optionally can be substituted with an alkylgroup. Examples of rings formed by substituents taken together with thenitrogen atom include, but are not limited to, morpholinyl,phenylpiperazinyl, imidazolyl, pyrrolidinyl, (N-methyl)-piperazinyl,piperidinyl, and the like.

[0055] The term “aryl” as used herein refers to a monocyclic, fusedbicyclic, and fused tricyclic carbocyclic ring systems having one ormore aromatic rings including, but not limited to, phenyl, naphthyl,tetrahydronaphthyl, phenanthrenyl, biphenylenyl, indanyl, indenyl,anthracenyl, phenanthrenyl, fluorenyl, and the like.

[0056] The term “carbamoyl” as used herein refers to a group of theformula —NR^(b)C(═O)R^(b), —OC(═O)N(R^(b)) and —NR^(b)C(═O)—, whereinR^(b) is hydrogen, alkyl, substituted alkyl (including trifluoromethyl),aryl, substituted aryl, heteroaryl, or substituted heteroaryl.

[0057] The term “carbonyl” as used herein refers to a —(CO)— (or —C═O)group.

[0058] The term “carboxyl” as used herein refers to —CO₂H.

[0059] The term “carboxamide” as used herein refers to —C(═O)N(R^(c))₂,wherein R^(c) is defined as hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic,substituted heterocyclic, cycloalkyl, and OR^(h), or the R^(c) groupsare taken together with the nitrogen to which they are attached to forma five- or six-membered optionally substituted aromatic or nonaromaticring, wherein one or two carbons of the ring optionally are replacedwith a heteroatom selected from the group consisting of sulfur, oxygen,and nitrogen.

[0060] The term “thiocarboxamide” as used herein refers to—C(═S)N(R^(c))₂, wherein R^(c) is defined above.

[0061] The term “mercapto” as used herein refers to —SR^(c), whereinR^(c) is defined above.

[0062] The term “sulfonamido” as used herein refers to —NHSO₂R^(c),wherein R^(c) is defined above.

[0063] The term “sulfo” as used herein refers to —SO₃H, and halidederivatives thereof, like —SO₂Cl, i.e., sulfonyl chloride.

[0064] The term “carboxy” as used herein refers to a —COOR^(a), whereinR^(a) is defined above.

[0065] The term “cyano” as used herein refers to a C═N group, alsodesignated —CN.

[0066] The term “hydroxyamino” as used herein refers to a —NHOH group.

[0067] The term “hydrazono” as used herein refers to a ═N—NH₂ group,wherein one or both hydrogen atoms can be replaced with an alkyl orsubstituted alkyl group.

[0068] The terms “trifluoromethyl” and “trifluoromethoxy” as used hereinrefer to —CF₃ and —OCF₃, respectively.

[0069] The term “halo” as used herein refers to bromo, chloro, iodo, andfluoro.

[0070] The term “phosphate” as used herein refers to a —OP(═O)(OR^(c))₂,and salts thereof.

[0071] The term “sulfonyl” as used herein refers to group represented by—SO₂— or —SO₂—R^(a) wherein R^(a) is defined above.

[0072] The term “sulfamyl” as used herein refers to —SO₂N(R^(c))₂,wherein R^(c) is defined above.

[0073] The term “nitro” as used herein refers to —NO₂.

[0074] The term “heteroaryl” as used herein refers to a cyclic aromaticring system having five to ten ring atoms, wherein one to four-ringatoms is selected from the group consisting of oxygen, nitrogen, andsulfur and the remaining ring atoms are carbon, said ring system beingjoined to the remainder of the molecule by any of the ring atoms.Nonlimiting examples of heteroaryl include pyridyl, pyrazinyl,pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, tetrazolyl,oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl,quinolinyl, isoquinolinyl, benzoxazolyl, benzimidazolyl, benzothiazolyl,and the like.

[0075] The term “heterocycloalkyl,” and “heterocyclic” as used hereinrefers to a nonaromatic partially unsaturated or fully saturated 3- to10-membered ring system, which includes single rings of 3 to 8 atoms,and bi- or tri-cyclic ring systems. These heterocyclic ring systemsinclude those having from one to four heteroatoms independently selectedfrom oxygen, nitrogen, and sulfur, wherein the nitrogen and sulfurheteroatoms optionally can be oxidized and the nitrogen heteroatomoptionally can be substituted. Representative heterocyclics include, butare not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl,imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl,isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl,tetrahydrofuryl, and the like.

[0076] The term “substituted aryl,” “substituted heteroaryl,” and“substituted heterocyclic” as used herein refer to an aryl, heteroaryl,or heterocyclic group substituted by a replacement of one, two, or threeof the hydrogen atoms thereon with a substitute selected from the groupconsisting of halo, OR^(h), N(R^(h))₂, CN, alkyl, substituted alkyl,mercapto, nitro, aldehyde, carboxy, carboxyl, carboxamide, aryl,heteroaryl, cycloalkyl, heterocyclic, O(CH₂)₁₋₃N(R^(h))₂, O(CH₂)₁₋₃CO₂H,and trifluoromethyl.

[0077] DNA-PK Inhibitors

[0078] The invention provides compounds that inhibit DNA-PK biologicalactivity. Inhibitor compounds of the present invention have a formula(I) or (II). Preferred compounds are those of formula (I) and (II)wherein A is a morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl,or tetrahydropyranyl group and L is absent. Other preferred DNA-PKinhibitor compounds are those compounds of formula (I) and (II),wherein:

[0079] n is an integer from 0 through 4;

[0080] Z, independently, is CR³ or N, or CR⁷ or N;

[0081] L is absent, or L is selected from the group consisting of—(CH₂)_(p)—, —(CHR^(k))_(p)—, —NR^(k)—(CHR^(k))_(p)—,—(CHR^(k))—NR^(k)—, —NR^(k)—, —C(═O)—, —O—, —NR^(k)—(CO)—,—(CO)—NR^(k)—, —S—, —SO—, —SO₂—, and —NR^(s)R^(t) (only if A is absent),wherein p is an integer 1 to 5;

[0082] R^(k) is selected from the group consisting of alkyl, aryl, andhydrogen;

[0083] R^(s) is selected from the group consisting of hydrogen, andalkyl;

[0084] R^(t) is alkyl, optionally substituted with oxo, hydroxy,methoxy, benzyloxy, halo, aryl, or heteroaryl;

[0085] A is absent, or is selected from the group consisting of a four-to seven- membered heterocyclic ring containing 1 or 2 heteroatomsindependently selected from the group consisting of N, O, and S;

[0086] R¹ or R⁵ is selected from the group consisting of —H, —NH₂,—(CO)—NH₂, —(CO)—NH—OH, —(CO)—NH—NH₂, —(CO)—NH—NH—R^(f), —(CO)—OH,—(CO)—O—CH₃, —(CO)—O—CH₂—CH₃, —(CO)-(4-methoxy)phenyl,—(CO)-(4-hydroxy)phenyl, —(CO)-(3-chlorophenyl), —(CO)-phenyl,—(CO)-benzyl, —(CO)—C₁₋₄alkyleneOR^(h), —(CO)—C₁₋₄alkyleneSR^(h),

[0087] —NO₂, —OH, —(CO)—C₁₋₄ alkyl, -cycloalkyl, —(CO)-substitutedalkyl, —(CO)-(methoxy)alkyl, —(CO)-(alkoxy) substituted alkyl,—(CO)-aryl, —(CO)-heteroaryl, —(CO)-(substituted alkyl)_(p)-aryl,—(CO)-(substituted alkoxy)_(p)-aryl, —(CO)—((NR^(k))_(p)-substitutedalkoxy)-aryl, —(CO)-aryl-R^(d), —(CO)-aryl-R^(e), —(CO)-aryl-R^(f),—CH═N—OH, —CH═N—NH₂, —CH═N—NH—CH₃, —CH═N—NH—CH₂-phenyl, —CF₃, —(CO)—CF₃,—(CO)—CH₂-morpholinyl, —(CO)—CH₂-heteroaryl, —(CO)—CH₂—CH—(CH₃)₂,—(CO)—CH₂—CH₂—(SO₂)—CH₃, —CHO, —C≡N, —CH₂—OH, —(CO)NR^(d)R^(e),—(CS)—NH₂, —(CO)—R^(f), —(CO)—CH₂Cl, —(CO)—CH₂—NR^(d)R^(e),—(CO)—CH₂—S—(CO)—CH₃, —(CO)—CH₂—SH, —(SO₂)-phenyl,2-(anilino)-4-thiazolyl-, 2-(pyridyl)-4-thiazolyl-, -benzoxazolyl,-imidazolyl, -thiazolyl, -substituted thiazolyl, -benzimidazolyl,-benzothiazolyl, -tetrazolyl, -(N-benzyl)-tetrazolyl,-(N-methyl)-tetrazolyl, -pyrazolyl, -(N-benzyl)-pyrazolyl,-(N-methyl)-pyrazolyl, -(N-acetyl)-pyrazolyl, -(N-mesyl)-pyrazolyl,-pyrazolyl-(CO)R^(u)R^(v), -(N-phenyl)-piperazinyl, -isoxazolyl,-pyrimidinyl, -(2-NH—CH₂-phenyl)-pyrimidinyl,-(2-(SO)-methyl)-pyrimidinyl,-(2-N-(N-t-butoxycarbonyl)-piperazinyl)-pyrimidinyl, and-(2-NH—CH₂-pyridine)-pyrimidinyl;

[0088] wherein R^(d) is selected from the group consisting of —H,-alkyl, —CH₂-phenyl, -phenyl, —O—CH₃, -pyridyl, -thiazolyl, -thiazinyl,—O—CH₂-pheny, —O-phenyl, —O-methoxyphenyl, —OH, —CH₂—(CO)—O—CH₃, and—CH₂—(CO)—OH; and

[0089] R^(e) is selected from the group consisting of —H,—CH₂—CH₂—O—CH₃, —CH₂—CH₂—CH₂—N(CH₃)₂, —O—CH₃, —CH₂—CH₂—(SO₂)—CH₃,—O—CH₃, —CH₂-pyridyl, —CH₂-phenyl, -alkyl, —CH₂—(CO)—O—CH₃, and-cylcopropyl; or

[0090] R^(d) and R^(e) are taken together to form -morpholinyl,-phenylpiperazinyl, -imidazolyl, -pyrrolidinyl, -(N-methyl)-piperazinyl,and -piperidinyl;

[0091] R^(f) is selected from the group consisting of -phenyl,-phenyl-(CF₃), -methylphenyl, -methoxyphenyl, -pyridyl, -alkyl, -benzyl,-thiophenyl, -thiazolyl, -chlorophenyl, -C(═NH)—NH₂, -fluorophenyl,—(CO)-phenyl, -(CH₂)-phenyl;

[0092] R^(u) is selected from the group consisting of —H, and -alkyl;

[0093] R^(v) is selected from the group consisting of —O—(CO)—CH₃,—NH—t-butoxycarbonyl, —O-phenyl, and —O—CH₂-phenyl; or

[0094] R^(u) and R^(v) are taken together with the carbon atoms to whichthey are attached to form a 5-membered ring containing an N, said Noptionally protected with t-butoxycarbonyl,

[0095] R² or R⁶ is selected from the group consisting of —H, —OH, -Halo,—CH₂—OH, —(CO)—NH₂, —NH₂, —(CO)—O—CH₃, —O—CH₃, —NH—(CO)—CF₃,—NH—(CO)—CH₃, —NH—(SO₂)—CH₃, —NH—CH₃, —N(CH₃)—(CO)—CF₃,—N═((CH(phenyl)-CH₂—(CO)OH, —NO₂, —O—PO₃ ^(═), —O-alkyl,—O—(CH₂)_(p)—OH, —O—(CH₂)_(p)—O-benzyl, —O—(CO)-heteroaryl,—O—(CO)-amino acid, —O—(CO)-nicotinic acid, —O—(CO)-aryl, —O—(CO)-alkyl,—O—CH₂—(CO)-benzyl, —O—(SO₂)—O—CF₃, —(CH₂)—CH═CH═N(CH₃)₂, —O—(SO₃)—, and—O—(PO)(OR^(j))(OR^(k));

[0096] wherein R^(j) independently are H, aryl, alkyl, or heterocyclic;or

[0097] R¹ and R², or R⁵ and R⁶, are taken together to form a three- orfour- membered component, respectively, of a five- or six-membered ring,preferably said ring selected from the group consisting of-2-imidazolidonyl-, -R^(g)- thiazolyl-, -carbonylpyrrolyl methylketone-, -4-imino-1,3,2,-oxathiaphosphanyl-2-thione-, -4-imino-1,3,2,-oxathiaphosphanyl-2-thione-2-(4′-methoxy)-phenyl-,-3-oxofuranyl-, -N-acetyl-3 -oxopyrrolinyl-, —N—(CH₂—COOH)-quinolonyl-,-N-(t-butoxycarbonyl)-quinolonyl-, —N—(CH₂—COOH)-quinolinyl-,—N—(t-butoxycarbonyl)-quinolinyl-, and

[0098] wherein B is aryl or a nitrogen-containing heteroaryl, R⁹ is H orOR^(h), and R¹⁰ is selected from the group consisting of halo, OR^(h),O(CH₂)₁₋₃N(R^(h))₂, O(CH₂)₁₋₃CO₂H, CN, morpholinyl, andN-(4-methyl)-piperazinyl,

[0099] wherein R^(g) is selected from the group consisting of -pyridyland -anilino;

[0100] R³ or R⁷, independently, is selected from the group consisting of—H, —OH, —OR^(d), —NO₂, —NH₂, —NH—R^(d), -halo, —CHO, —(SO₂)—OH,-(SO₂)—Cl, and —(SO₂)—NR^(i)R^(k);

[0101] wherein R¹ is selected from the group consisting of —H, —CH₃,—CH₂phenyl, -phenyl, —CH₂—CH₂—O—CH₃, —CH₂—CH₂—CH₂—N(CH₃)₂, —O—CH₃,—CH₂—CH₂—(SO₂)—CH₃, -pyridyl, -thiazolyl, —O—CH₂-phenyl, —OH,—CH₂—(CO)—O—CH₃, and —CH₂—(CO)—OH;

[0102] R^(k) is selected from the group consisting of —H, —O—CH₃,—CH₂-pyridyl, —CH₂-phenyl, —CH₃, —CH₂—(CO)—O—CH₃, -cyclopropyl, and—CH₂-cyclopropyl; or

[0103] R^(i) and R^(k)are taken together to form morpholinyl,phenylpiperazinyl, imidazolyl, pyrrolidinyl, (N-methyl)-piperazinyl, andpiperidinyl; and

[0104] R⁴ or R⁸, independently, is selected from the group consisting of—H, —CH₃, —OCH₃, —OH, —(CO)—CH₃, -methoxyphenyl, and -pyridinyl.

[0105] The preferred groups for the substituents R¹ and R⁵ in a compoundof formula (I) or (II) are —H, —OH, —NH₂, —CH₂OH, —C═N, —(CO)—NH₂,—(CO)—OH, —(CO)—O—CH₃, —CH═N—OH, —CH═N—NH₂, —CH═N—NH—CH₃,—(CO)—CF₃,—(CO)H, —NO₂, —(CO)-alkyl, —(CO)-substituted alkyl,—(CO)-aryl, —(CO)-substituted aryl, —(CO)-heteroaryl,—(CO)—CH₂—NR^(d)R^(e), and —(CO)NR^(d)R^(e), wherein R^(d) and R^(e) areas previously defined.

[0106] The preferred groups for the substituent R² and R⁶ in a compoundof formula (I) or (II) are —H, —OH, —F, —CH₂—OH, —NH₂, —NH—(CO)—CF₃,—NH—(CO)—CH₃, —NH—(SO₂)—CH₃, —NH—CH₃, and —N(CH₃)—(CO)—CF₃.

[0107] Examples of compounds of the invention include, but are notlimited to, compounds described in Table 1, below. TABLE 1 DNA-PKINHIBITORS Benzyl 2-((4-benzyl)carbonyl)-5-morpholin-4-yl-benzenephosphate 4-Methylphenyl 4-morpholin-4-yl-2-(phosphonooxy)phenylmethanone disodium salt 5-Morpholin-4-yl-2-nitrophenylamine5-(4-Methyl-piperazin-1-yl)-2-nitrophenylamine2-Hydroxymethyl-5-morpholin-4-yl-phenol2-Nitro-5-thiomorpholin-4-yl-phenylamineN¹-Morpholin-4-yl-4-nitrobenzene-1,3-diamine1-(3-Amino-4-nitrophenyl)-piperidin-4-ol2-Nitro-5-piperidin-1-yl-phenylamine5-(4-acetylpiperazin-1-yl)-2-nitrophenylamine2-Nitro-5-piperazin-1-yl-phenylamine1-(3-Amino-4-nitrophenyl)-piperidin-3-olN¹-(2-Morpholin-4-yl-ethyl)-4-nitrobenzene-1,3-diamine5-[4-(2-Methoxyphenyl)-piperazin-1-yl]-2-nitrophenylamine5-(cis-2,6-Dimethylmorpholin-4-yl)-2-nitrophenylamine2-Nitro-5-(4-pyridin-2-yl-piperazin-1-yl)-phenylamineN¹-(3-Morpholin-4-yl-propyl)-4-nitrobenzene-1,3-diamine2-Hydroxy-4-morpholin-4-yl-benzonitrile(5-Morpholin-4-yl-2-nitrophenyl)-methanol2-Hydroxy-4-morpholin-4-yl-benzoic acid2-Hydroxy-4-morpholin-4-yl-benzoic acid methyl ester5-Morpholin-4-yl-2-nitro-benzamide2-Hydroxy-4-morpholin-4-yl-benzaldehyde 5-Morpholin-4-yl-2-nitro-phenol1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-ethanone1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-propan-1-one1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-3-methyl-butan-1-one1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-phenyl-methanone2,2,2-Trifluoro-1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone4-Amino-2-morpholin-4-yl-pyrimidine-5-carboxylic acid1-(5-Bromo-2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone1-(3-Bromo-2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone1-(3,5-Dichloro-2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone1-(3-Chloro-2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone1-(5-Fluoro-2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone1-(3-Fluoro-2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone1-[2-Hydroxy-4-(tetrahydropyran-4-yloxy)-phenyl]-ethanone5-(Morpholin-4-yl)-1,3-dihydro-benzimidazol-2-one2-Methoxy-4-morpholin-4-yl-benzaldehyde4-Methoxy-6-morpholin-4-yl-benzene-1,3-dicarbaldehyde2-Hydroxy-5-morpholin-4-yl-benzoic acid methyl ester2-((Hydroxyimino)methyl)-5-morpholin-4-yl-phenol2-Hydrazonomethyl-5-morpholin-4-yl-phenol2-Hydroxy-4-[(1-morpholin-4-yl-methanoyl)amino]-benzoic acid2-Hydroxy-4-morpholin-4-ylmethyl-benzoic acid methyl ester hydrochloride2-Hydroxy-4-morpholin-4-ylmethyl-benzoic acid trifluoroacetate2-Hydroxy-4-morpholin-4-ylmethyl benzoic acid hydrochloride4-Amino-2-hydroxy-benzoic acid methyl ester2-Hydroxy-4-morpholin-4-yl-benzoic acid methyl ester2-Hydroxy-N-methyl-4-morpholin-4-yl-benzamide1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-morpholin-4-yl-methanone2-Hydroxy-4-morpholin-4-yl-benzamide2-Hydroxy-4-morpholin-4-yl-N-benzyl-benzamide2-Hydroxy-4-morpholin-4-yl-N-phenyl-benzamideN-Cyclopropyl-2-hydroxy-4-morpholin-4-yl-N-phenyl-benzamide2-Hydroxy-N-(2-methoxyethyl)-4-morpholin-4-yl-benzamide2-Hydroxy-4-morpholin-4-yl-N-methoxy-N-methyl-benzamide2-Hydroxy-4-morpholin-4-yl-N-(3-dimethylaminopropyl)-benzamide2-Hydroxy-N-methoxy-4-morpholin-4-yl-benzamide2-Hydroxy-N-(2-methanesulfonylethyl)-4-morpholin-4-yl-benzamide2-Hydroxy-4-morpholin-4-yl-N-pyridin-3-yl-benzamide2-Hydroxy-4-morpholin-4-yl-N-pyridin-4-yl-benzamide2-Hydroxy-4-morpholin-4-yl-N-thiazol-2-yl-benzamide2-Hydroxy-4-morpholin-4-yl-N-(1,4-thiazin-2-yl)-benzamide2,N-Dihydroxy-4-morpholin-4-yl-benzamide2-Hydroxy-4-morpholin-4-yl-N-(4-pyridylmethyl)-benzamide1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-(4-phenylpiperizin-1-yl)-methanone 2-Hydroxy-4-morpholin-4-yl-benzoic acidN-Carboxymethyl-2-hydroxy-4-morpholin-4-yl-phenyl)-carboxamide methylester N-Carboxymethyl-2-hydroxy-4-morpholin-4-yl-phenyl-carboxamide2-Hydroxy-4-morpholin-4-yl-thiobenzamide2-(4-Ethylphenyl)-4-imino-7-morpholin-4-yl-benzo(e]-1,3,2-oxathiaphosphane-2-thione1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-phenyl-methanone1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-(4-trifluoromethylphenyl)-methanone 1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-(o-tolyl)-methanone1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-(4-methoxyphenyl)-methanone1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-pyridin-3-yl-methanone1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-pentan-1-one1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-2-phenyl-ethanone1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-thiophen-2-yl-methanone2-Hydroxy-4-morpholin-4-yl-phenyl-1,3-thiazol-2-yl ketone1-(3-Chlorophenyl)-1-(2-hydroxy-4-morpholin-4-yl-phenyl)-methanone2-Chloro-1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-2-morpholin-4-yl-ethanone1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-2-imidazol-1-yl-ethanone1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-pyrrolidin-1-yl-methanone1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-(4-methyl-piperazin-1-yl)-methanone 2-Hydroxy-4-morpholin-4-yl-phenyl-1-piperidin-1-yl-methanone2-(Benzyl-methyl-amino)-1-(2-hydroxy-4-morpholin-4-yl-phenyl)- ethanone2-Acetylthio-1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-2-mercapto-ethanone6-Morpholin-4-yl-2-hydrobenzo(b]furan-3-one4-[2-Methyl-4-morpholin-4-yl-phenyl)-2-(3-pyridyl)-1,3-thiazole5-Morpholin-4-yl-2-(2-phenylamino-1,3-thiazol-4-yl)-phenol3-Methoxy-1-morpholin-4-yl-benzene4-Methoxy-2-morpholin-4-yl-benzenesulfonic acid4-Methoxy-2-morpholin-4-yl-benzenesulfonyl chloride4-Methoxy-N-methyl-2-morpholin-4-yl-benzenesulfonamide4-Methoxy-2-morpholin-4-yl-N-benzyl-benzenesulfonamide4-Methoxy-2-morpholin-4-yl-N-cyclopropylmethyl-benzenesulfonamideN,N-Diethyl-(3-morpholin-4-yl-phenoxy)carboxamideN,N-Diethyl-(2-benzenesulfonyl-5-morpholin-4-yl-phenoxy)carboxamide2-Benzenesulfonyl-5-morpholin-4-yl-phenol3-Nitro-1-morpholin-4-yl-benzene 3-Morpholin-4-yl-phenylamine1-(2-Amino-4-morpholin-4-yl-phenyl)-2-chloro-ethanone2-Amino-4-morpholin-4-yl-N-benzyl-N-methyl-benzamide1-(2-Amino-4-morpholin-4-yl-phenyl)-1-pyrrolidin-1-yl-methanone(2-Amino-4-morpholin-4-yl-phenyl)-1-piperidin-1-yl-methanone2-Amino-4-fluorobenzoic acid methyl ester4-Fluoro-2-(2,2,2-trifluoroacetylamino)-benzoic acid methyl ester4-Morpholin-4-yl-2-(2,2,2-trifluoroacetylamino)-benzoic acid methylester 2-Amino-4-morpholin-4-yl-benzoic acid2-Methylsulfonylamino-4-morpholin-4-yl-benzoic acid4-Morpholin-4-yl-2-(2,2,2-trifluoroacetylamino)-N-benzyl-benzamideN,N-Dimethyl-4-morpholin-4-yl-2-(2,2,2-trifluoroacetylamino)-benzamide2-Amino-4-morpholin-4-yl-N,N-dimethyl-benzamideN-Methyl-4-morpholin-4-yl-2-(2,2,2-trifluoroacetylamino)-benzamide2-Amino-4-morpholin-4-yl-benzoic acid methyl ester2-Acetylamino-4-morpholin-4-yl-benzoic acid methyl ester2-Acetylamino-4-morpholin-4-yl-benzoic acid2-Methanesulfonylamino-4-morpholin-4-yl-benzoic acid methyl ester(2-N-Methyl-N-(2,2,2-trifluoroacetyl)amino)-4-morpholin-4-yl-benzoicacid methyl ester 2-Methylamino-4-morpholin-4-yl-benzoic acid methylester 2-Methylamino-4-morpholin-4-yl-benzoic acid2-Chloro-1-(2-acetamido-4-morpholin-4-yl-phenyl)-ethanone1-Acetyl-6-morpholin-4-yl-1,2-dihydro-indol-3-one4-Morpholin-4-yl-2-nitro-benzoic acid methyl ester4-Morpholin-4-yl-2-nitro-benzoic acid4-Morpholin-4-yl-2-nitrophenyl)-N-(methylcarboxymethyl)benzamide5-Hydroxy-7-morpholin-4-yl-2-phenyl-chromen-4-one5-Hydroxy-2-phenyl-7-piperidin-1-yl-chromen-4-oneTrifluoromethanesulfonic acid 3,5-dihydroxy-4-oxo-2-phenyl-4H-chromen-7-yl ester3,5-Dihydroxy-7-morpholin-4-yl-2-phenyl-chromen-4-oneTrifluoromethanesulfonic acid 4-acetyl-3,5-dihydroxy-phenyl ester1-(2,6-Dihydroxy-4-morpholin-4-yl-phenyl)-ethanone4-(5-Hydroxy-7-morpholin-4-yl-4-oxo-4H-chromen-2-yl)-benzonitrile3-(5-Hydroxy-7-morpholin-4-yl-4-oxo-4H-chromen-2-yl)-benzonitrile5-Hydroxy-2-(4-methoxyphenyl)-7-morpholin-4-yl-Chromen-4-one5-Hydroxy-7-morpholin-4-yl-2-pyridin-3-yl-chromen-4-one2-Hydroxy-1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone2-Hydroxy-1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone.

[0108] DNA-PK inhibitor compounds of the invention can exist asstereoisomers having asymmetric or chiral centers. Stereoisomers aredesignated by either “S” or “R” depending on arrangement of substituentsaround a chiral carbon atom. Mixtures of stereoisomers are contemplatedunder the invention. Stereoisomers include enantiomers, diastereomers,and mixtures of the two. Individual stereoisomers of compounds of theinvention can be prepared synthetically from commercially availablestarting materials which contain asymmetric or chiral centers or bypreparation of racemic mixtures followed by separation or resolutiontechniques well known in the art. Methods of resolution include (1)attachment of a mixture of enantiomers to a chiral auxiliary, separationof the resulting mixture by recrystallization or chromatography, andliberation of the optically pure product from the auxiliary, (2) saltformation employing an optically active resolving agent, and (3) directseparation of the mixture of optical enantiomers on chiralchromatographic columns.

[0109] The invention also provides prodrug forms of DNA-PK inhibitors ofthe invention. Prodrug design is discussed generally in Hardma et al.,(Eds), Goodman & Gilman's The Pharmacological Basis of Therapeutics,Ninth Edition, New York, N.Y. (1996), pp. 11-16. Briefly, administrationof a drug is followed by elimination from the body or somebiotransformation whereby biological activity of the drug is reduced oreliminated. Alternatively, a biotransformation process can lead to ametabolic by-product which is itself more active or equally active ascompared to the drug initially administered. Increased understanding ofthese biotransformation processes permits the design of so-called“prodrugs” which, following a biotransformation, become morephysiologically active in an altered state. Prodrugs arepharmacologically inactive or active compounds which are converted tobiologically active or more active metabolites. In some forms, prodrugsare rendered pharmacologically active through hydrolysis of, forexample, an ester or amide linkage, often times introducing or exposinga functional group on the prodrug. The thus modified drug also can reactwith an endogenous compound to form a water soluble conjugate whichfurther increases pharmacological properties of the compound, forexample, as a result of increased circulatory half-life.

[0110] As another alternative, prodrugs can be designed to undergocovalent modification on a functional group with, for example,glucuronic acid, sulfate, glutathione, amino acids, or acetate. Theresulting conjugate can be inactivated and excreted in the urine, orrendered more potent than the parent compound. High molecular weightconjugates also can be excreted into the bile, subjected to enzymaticcleavage, and released back into circulation, thereby effectivelyincreasing the biological half-life of the originally administeredcompound. Prodrugs are particularly useful for delivering a compound toa predetermined site of action, and modifications can be effected tofacilitate targeting in this manner.

[0111] Pharmaceutical Compositions

[0112] The invention also provides pharmaceutical compositionscomprising one or more DNA-PK inhibitors of formula (I) or (II). Thepharmaceutical composition comprises the DNA-PK inhibitors of formula(I) or (II) in a pharmaceutically acceptable carrier or diluent,including, but not limited to, preferred compounds of formula (I) or(II). The invention also provides pharmaceutical compositions having acompound of formula (I) or (II) in combination with an anti-neoplasticagent.

[0113] In a preferred embodiment, the pharmaceutical compositionscomprise one or more DNA-PK inhibitor compounds including, but notlimited to, the compounds set forth below: TABLE 24-(4-(1-Phenylvinyl)phenyl]morpholine 4-(4-Nitrophenyl)morpholine Methyl7-amino-2-morpholin-4-yl-7a-hydro-1,2,4-triazolo(1,5-apyrimidine-6-carboxylate 1-(4-Morpholin-4-ylphenyl)ethan-1-oneBenzyl 2-[(4-benzyl)carbonyl]-5-morpholin-4-yl-benzene phosphate4-Methylphenyl 4-morpholin-4-yl-2-(phosphonooxy)phenyl methanonedisodium salt 5-Morpholin-4-yl-2-nitrophenylamine5-(4-Methylpiperazin-1-yl)-2-nitrophenylamine2-Hydroxymethyl-5-morpholin-4-yl-phenol2-Nitro-5-thiomorpholin-4-yl-phenylamineN¹-Morpholin-4-yl-4-nitrobenzene-1,3-diamine1-(3-Amino-4-nitrophenyl)-piperidin-4-ol2-Nitro-5-piperidin-1-yl-phenylamine5-(4-acetylpiperazin-1-yl)-2-nitrophenylamine2-Nitro-5-piperazin-1-yl-phenylamine1-(3-Amino-4-nitrophenyl)-piperidin-3-olN¹-(2-Morpholin-4-yl-ethyl)-4-nitrobenzene-1,3-diamine5-[4-(2-Methoxyphenyl)piperazin-1-yl]-2-nitrophenylamine5-(cis-2,6-Dimethylmorpholin-4-yl)-2-nitrophenylamine2-Nitro-5-(4-pyridin-2-yl-piperazin-1-yl)-phenylamineN¹-(3-Morpholin-4-yl-propyl)-4-nitrobenzene-1,3-diamine2-Hydroxy-4-morpholin-4-yl-benzonitrile(5-Morpholin-4-yl-2-nitrophenyl)-methanol2-Hydroxy-4-morpholin-4-yl-benzoic acid2-Hydroxy-4-morpholin-4-yl-benzoic acid methyl ester5-Morpholin-4-yl-2-nitro-benzamide2-Hydroxy-4-morpholin-4-yl-benzaldehyde 5-Morpholin-4-yl-2-nitro-phenol1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-ethanone1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-propan-1-one1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-3-methyl-butan-1-one1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-phenyl-methanone2,2,2-Trifluoro-1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone4-Amino-2-morpholin-4-yl-pyrimidine-5-carboxylic acid1-(5-Bromo-2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone1-(3-Bromo-2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone1-(3,5-Dichloro-2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone1-(3-Chloro-2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone1-(5-Fluoro-2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone1-(3-Fluoro-2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone1-[2-Hydroxy-4-(tetrahydropyran-4-yloxy)-phenyl]-ethanone5-(Morpholin-4-yl)-1,3-dihydro-benzimidazol-2-one2-Methoxy-4-morpholin-4-yl-benzaldehyde4-Methoxy-6-morpholin-4-yl-benzene-1,3-dicarbaldehyde2-Hydroxy-5-morpholin-4-yl-benzoic acid methyl ester2-((Hydroxyimino)methyl)-5-morpholin-4-yl-phenol2-Hydrazonomethyl-5-morpholin-4-yl-phenol2-Hydroxy-4-[(1-morpholin-4-yl-methanoyl)-amino]-benzoic acid2-Hydroxy-4-morpholin-4-ylmethyl-benzoic acid methyl ester hydrochloride2-Hydroxy-4-morpholin-4-ylmethyl-benzoic acid trifluoroacetate2-Hydroxy-4-morpholin-4-ylmethyl benzoic acid hydrochloride4-Amino-2-hydroxy-benzoic acid methyl ester2-Hydroxy-4-morpholin-4-yl-benzoic acid methyl ester2-Hydroxy-N-methyl-4-morpholin-4-yl-benzamide1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-morpholin-4-yl-methanone2-Hydroxy-4-morpholin-4-yl-benzamide2-Hydroxy-4-morpholin-4-yl-N-benzyl-benzamide2-Hydroxy-4-morpholin-4-yl-N-phenyl-benzamideN-Cyclopropyl-2-hydroxy-4-morpholin-4-yl-N-phenyl-benzamide2-Hydroxy-N-(2-methoxyethyl)-4-morpholin-4-yl-benzamide2-Hydroxy-4-morpholin-4-yl-N-methoxy-N-methyl-benzamide2-Hydroxy-4-morpholin-4-yl-N-(3-dimethylaminopropyl)-benzamide2-Hydroxy-N-methoxy-4-morpholin-4-yl-benzamide2-Hydroxy-N-(2-methanesulfonylethyl)-4-morpholin-4-yl-benzamide2-Hydroxy-4-morpholin-4-yl-N-pyridin-3-yl-benzamide2-Hydroxy-4-morpholin-4-yl-N-pyridin-4-yl-benzamide2-Hydroxy-4-morpholin-4-yl-N-thiazol-2-yl-benzamide2-Hydroxy-4-morpholin-4-yl-N-(1,4-thiazin-2-yl)-benzamide2,N-Dihydroxy-4-morpholin-4-yl-benzamide2-Hydroxy-4-morpholin-4-yl-N-(4-pyridylmethyl)-benzamide1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-(4-phenylpiperizin-1-yl)-methanone 2-Hydroxy-4-morpholin-4-yl-benzoic acidN-Carboxymethyl-2-hydroxy-4-morpholin-4-yl-phenyl)-carboxamide methylester N-Carboxymethyl-2-hydroxy-4-morpholin-4-yl-phenyl-carboxamide2-Hydroxy-4-morpholin-4-yl-thiobenzamide2-(4-Ethylphenyl)-4-imino-7-morpholin-4-yl-benzo(e)-1,3,2-oxathiaphosphane-2-thione1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-phenyl-methanone1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-(4-trifluoromethyl-phenyl)-methanone 1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-(o-tolyl)-methanone1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-(4-methoxyphenyl)-methanone1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-pyridin-3-yl-methanone1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-pentan-1-one1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-2-phenyl-ethanone1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-thiophen-2-yl-methanone2-Hydroxy-4-morpholin-4-yl-phenyl-1,3-thiazol-2-yl ketone1-(3-Chlorophenyl)-1-(2-hydroxy-4-morpholin-4-yl-phenyl)-methanone2-Chloro-1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-2-morpholin-4-yl-ethanone1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-2-imidazol-1-yl-ethanone1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-pyrrolidin-1-yl-methanone1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-(4-methylpiperazin-1-yl)-methanone 2-Hydroxy-4-morpholin-4-yl-phenyl-1-piperidin-1-yl-methanone2-(Benzyl-methyl-amino)-1-(2-hydroxy-4-morpholin-4-yl-phenyl)- ethanone2-Acetylthio-1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-2-mercapto-ethanone6-Morpholin-4-yl-2-hydrobenzo(b]furan-3-one4-[2-Methyl-4-morpholin-4-yl-phenyl)-2-(3-pyridyl)]-1,3-thiazole5-Morpholin-4-yl-2-(2-phenylamino-1,3-thiazol-4-yl)-phenol3-Methoxy-1-morpholin-4-yl-benzene4-Methoxy-2-morpholin-4-yl-benzenesulfonic acid4-Methoxy-2-morpholin-4-yl-benzenesulfonyl chloride4-Methoxy-N-methyl-2-morpholin-4-yl-benzenesulfonamide4-Methoxy-2-morpholin-4-yl-N-benzyl-benzenesulfonamide4-Methoxy-2-morpholin-4-yl-N-cyclopropylmethyl-benzenesulfonamideN,N-Diethyl-(3-morpholin-4-yl-phenoxy)carboxamideN,N-Diethyl-(2-benzenesulfonyl-5-morpholin-4-yl-phenoxy)carboxamide2-Benzenesulfonyl-5-morpholin-4-yl-phenol3-Nitro-1-morpholin-4-yl-benzene 3-Morpholin-4-yl-phenylamine1-(2-Amino-4-morpholin-4-yl-phenyl)-2-chloro-ethanone2-Amino-4-morpholin-4-yl-N-benzyl-N-methyl-benzamide1-(2-Amino-4-morpholin-4-yl-phenyl)-1-pyrrolidin-1-yl-methanone(2-Amino-4-morpholin-4-yl-phenyl)-1-piperidin-1-yl-methanone2-Amino-4-fluorobenzoic acid methyl ester4-Fluoro-2-(2,2,2-trifluoroacetylamino)-benzoic acid methyl ester4-Morpholin-4-yl-2-(2,2,2-trifluoroacetylamino)-benzoic acid methylester 2-Amino-4-morpholin-4-yl-benzoic acid2-Methylsulfonylamino-4-morpholin-4-yl-benzoic acid4-Morpholin-4-yl-2-(2,2,2-trifluoroacetylamino)-N-benzyl-benzamideN,N-Dimethyl-4-morpholin-4-yl-2-(2,2,2-trifluoroacetylamino)-benzamide2-Amino-4-morpholin-4-yl-N,N-dimethyl-benzamideN-Methyl-4-morpholin-4-yl-2-(2,2,2-trifluoroacetylamino)-benzamide2-Amino-4-morpholin-4-yl-benzoic acid methyl ester2-Acetylamino-4-morpholin-4-yl-benzoic acid methyl ester2-Acetylamino-4-morpholin-4-yl-benzoic acid2-Methanesulfonylamino-4-morpholin-4-yl-benzoic acid methyl ester(2-N-Methyl-N-(2,2,2,-trifluoroacetyl)-amino]-4-morpholin-4-yl-benzoicacid methyl ester 2-Methylamino-4-morpholin-4-yl-benzoic acid methylester 2-Methylamino-4-morpholin-4-yl-benzoic acid2-Chloro-1-(2-acetamido-4-morpholin-4-yl-phenyl)-ethanone1-Acetyl-6-morpholin-4-yl-1,2-dihydro-indol-3-one4-Morpholin-4-yl-2-nitro-benzoic acid methyl ester4-Morpholin-4-yl-2-nitro-benzoic acid4-Morpholin-4-yl-2-nitrophenyl)-N-(methylcarboxymethyl)benzamide5-Hydroxy-7-morpholin-4-yl-2-phenyl-chromen-4-one5-Hydroxy-2-phenyl-7-piperidin-1-yl-chromen-4-oneTrifluoromethanesulfonic acid 3,5-dihydroxy-4-oxo-2-phenyl-4H-chromen-7-yl ester 3,5-Dihydroxy-7-morpholin-4-yl-2-phenyl-chromen-4-oneTrifluoromethanesulfonic acid 4-acetyl-3,5-dihydroxy-phenyl ester1-(2,6-Dihydroxy-4-morpholin-4-yl-phenyl)-ethanone4-(5-Hydroxy-7-morpholin-4-yl-4-oxo-4H-chromen-2-yl)-benzonitrile3-(5-Hydroxy-7-morpholin-4-yl-4-oxo-4H-chromen-2-yl)-benzonitrile5-Hydroxy-2-(4-methoxyphenyl)-7-morpholin-4-yl-chromen-4-one5-Hydroxy-7-morpholin-4-yl-2-pyridin-3-yl-chromen-4-one2-Hydroxy-1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone2-Hydroxy-1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone.

[0114] In one aspect, the pharmaceutical compositions comprise acompound of formula (I) or (II) and one or more antineoplastic agents.In a preferred embodiment, the composition comprises a chemotherapeuticagent, one or more radiotherapeutic agents, or a combination of one ormore chemotherapeutic and radiotherapeutic agents in a pharmaceuticallyacceptable carrier or diluent. Examples of anti-neoplastic agents,including chemotherapeutic and radiotherapeutic agents, suitable for theinvention include, but are not limited to, compounds described in Table3 below. TABLE 3 CHEMOTHERAPEUTIC AGENTS Alkylating agents Naturalproducts Miscellaneous agents Nitrogen mustards Antimitotic drugsPlatinium coordination complexes mechlorethamine paclitaxel cisplatincyclophosphamide Vinca alkaloids carboplatin ifosfamide vinblastine(VLB) Anthracenedione melphalan vincristine mitoxantrone chlorambucilvinorelbine Substituted urea Nitrosoureas Taxotere ® (docetaxel)hydroxyurea carmustine (BCNU) estramustine Methylhydrazine derivativeslomustine (CCNU) estramustine phosphate N-methylhydrazine (MIH)semustine (methyl-CCNU) Epipodophylotoxins procarbazineEthylenimine/Methylmelamine etoposide Adrenocortical suppressantthriethylenemelamine (TEM) teniposide mitotane (o,p′-DDD) triethylenethiophosphoramide Antibiotics aminoglutethimide (thiotepa) actimomycin DCytokines hexamethylmelamine daunomycin (rubidomycin) interferon (α, β,γ) (HMM, altretamine) doxorubicin (adriamycin) interleukin-2 Alkylsulfonates mitoxantrone Hormones and antagonists busulfan idarubicinAdrenocorticosteroids/antagonists Triazines bleomycins prednisone andequivalents dacarbazine (DTIC) plicamycin (mithramycin) dexamethasoneAntimetabolites mitomycinC aminoglutethimide Folic Acid analogsdactinomycin Progestins methotrexate Enzymes hydroxyprogesteronecaproate trimetrexate L-asparaginase medroxyprogesterone acetatePyrimidine analogs megestrol acetate 5-fluorouracil Biological responsemodifiers Estrogens fluorodeoxyuridine interferon-alphadiethylstilbestrol gemcitabine IL-2 ethynyl estradiol/equivalentscytosine arabinoside G-CSF Antiestrogen (AraC, cytarabine) GM-CSFtamoxifen 5-azacytidine Differentiation Agents Androgens2,2′-difluorodeoxycytidine retinoic acid derivatives testosteronepropionate Purine analogs Radiosensitizers fluoxymesterone/equivalents6-mercaptopurine metronidazole Antiandrogens 6-thioguanine misonidazoleflutamide azathioprine desmethylmisonidazole gonadotropin-releasing2′-deoxycoformycin pimonidazole hormone analogs (pentostatin)etanidazole leuprolide erythrohydroxynonyladenine nimorazoleNonsteroidal antiandrogens (EHNA) RSU 1069 flutamide fludarabinephosphate EO9 Photosensitizers 2-Chlorodeoxyadenosine RB 6145hematoporphyrin derivatives (cladribine, 2-CdA) SR4233 Photofrin ® TypeI Topoisomerase nicotinamide benzoporphyrin derivatives Inhibitors5-bromodeozyuridine Npe6 camptothecin 5-iododeoxyuridine tinetioporphyrin (SnET2) topotecan bromodeoxycytidine pheoboride-airinotecan bacteriochlorophyll-a naphthalocyanines

[0115] Depending on the neoplastic condition, pharmaceuticalcompositions of the invention can be formulated to include one or morecytokines, lymphokines, growth factors, or other hematopoietic factorswhich can reduce negative side effects that may arise from, or beassociated with, administration of the pharmaceutical composition alone.Cytokines, lymphokines, growth factors, or other hematopoietic factorsparticularly useful in pharmaceutical compositions of the inventioninclude, but are not limited to, M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14,IL-15, IL-16, IL-17, IL-18, IFN, TNF, G-CSF, Meg-CSF, GM-CSF,thrombopoietin, stem cell factor, erythropoietin, angiopoietins,including Ang-1, Ang-2, Ang-4, Ang-Y, and/or the human angiopoietin-likepolypeptide, vascular endothelial growth factor (VEGF), angiogenin, bonemorphogenic protein-1 (BMP-1), BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7,BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, BMPreceptor IA, BMP receptor IB, brain derived neurotrophic factor, ciliaryneutrophic factor, ciliary neutrophic factor receptor a cytokine-inducedneutrophil chemotactic factor 1, cytokine-induced neutrophil chemotacticfactor 2 α, cytokine-induced neutrophil chemotactic factor 2 β, βendothelial cell growth factor, endothelin 1, epidermal growth factor,epithelial-derived neutrophil attractant, fibroblast growth factor (FGF)4, FGF 5, FGF 6, FGF 7, FGF 8, FGF 8b, FGF 8c, FGF 9, FGF 10, FGFacidic, FGF basic, glial cell line-derived neutrophic factor receptor α1, glial cell line-derived neutrophic factor receptor α 2, growthrelated protein, growth related protein α, growth related protein β,growth related protein γ, heparin binding epidermal growth factor,hepatocyte growth factor, hepatocyte growth factor receptor,insulin-like growth factor I, insulin-like growth factor receptor,insulin-like growth factor II, insulin-like growth factor bindingprotein, keratinocyte growth factor, leukemia inhibitory factor,leukemia inhibitory factor receptor α, nerve growth factor nerve growthfactor receptor, neurotrophin-3, neurotrophin-4, placenta growth factor,placenta growth factor 2, platelet-derived endothelial cell growthfactor, platelet derived growth factor, platelet derived growth factor Achain, platelet derived growth factor AA, platelet derived growth factorAB, platelet derived growth factor B chain, platelet derived growthfactor BB, platelet derived growth factor receptor α, platelet derivedgrowth factor receptor β, pre-B cell growth stimulating factor, stemcell factor, stem cell factor receptor, transforming growth factor (TGF)α, TGF β, TGF β1, TGF β1.2, TGF β2, TGF β3, TGF β5, latent TGF β1, TGFβ, binding protein I, TGF β binding protein II, TGF β binding proteinIII, tumor necrosis factor receptor type I, tumor necrosis factorreceptor type II, urokinase-type plasminogen activator receptor,vascular endothelial growth factor, and chimeric proteins andbiologically or immunologically active fragments thereof.

[0116] The therapeutic index of compositions comprising one or moreincompounds of the invention can be enhanced by conjugation of thecompound(s) with anti-tumor antibodies as previously described (forexample, Pietersz and McKinzie, Immunol. Rev. 129:57 (1992); Trail etal., Science 261:212 (1993); Rowlinson-Busza and Epenetos, Curr. Opin.Oncol. 4:1142 (1992)). Tumor directed delivery of compounds of theinvention enhances the therapeutic benefit by minimizing potentialnonspecific toxicities which can result from radiation treatment orchemotherapy. In another aspect, DNA-PK inhibitor compounds andradioisotopes or chemotherapeutic agents can be conjugated to the sameantibody molecule. Alternatively, DNA-PK inhibitor-conjugated tumorspecific antibodies can be administered before, during, or afteradministration of chemotherapeutic-conjugated antitumor antibody orradioimmunotherapy.

[0117] Methods to Inhibit DNA-PK

[0118] The invention further provides methods of inhibiting DNA-PKactivity comprising the step of contacting DNA-PK or a biologicallyactive fragment thereof, with one or more compounds of formula (I) or(II). Particular examples of the compounds suitable for the methodinclude, but are not limited to, compounds as described in Table 2.Methods of the invention include in vivo, in vitro, and ex vivoapplications. Cells useful in the methods include those that expressendogenous DNA-PK enzymes, “endogenous” indicating that the cellsexpress DNA-PK absent recombinant introduction into the cells of one ormore polynucleotides encoding a DNA-PK enzyme or a biologically activefragment thereof. Methods also contemplate use of cells that expressexogenous DNA-PK wherein one or more polynucleotides encoding a DNA-PKenzyme or biologically active fragment thereof have been introduced intothe cell using recombinant procedures. In another aspect, methodsinclude use of cancer cells. In a preferred embodiment, methods includeuse of mammalian cancer cells, and in a most preferred method, themammalian cancer cells are human cancer cells.

[0119] In vitro methods also are contemplated comprising the step ofcontacting DNA-PK with an inhibitor of the invention. The DNA-PK enzymeof an in vitro method can include a purified and isolated enzyme,wherein the enzyme is isolated from natural sources (i.e., cells ortissues that normally express a DNA-PK enzyme absent modification byrecombinant technology) or isolated from cells modified by recombinanttechniques to express an exogenous enzyme.

[0120] Methods to Identify DNA-PK Inhibitors

[0121] The invention also provides methods of identifying DNA-PKinhibitors comprising the steps of a) measuring DNA-PK enzyme activityin the presence and absence of a test compound, and b) identifying thetest compound as a DNA-PK inhibitor when DNA-PK enzyme activity isdecreased in the presence of the test compound. The inventioncontemplates in vivo and in vitro methods. In one aspect, purified andisolated DNA-PK is utilized in the method. The enzyme can be obtainedfrom cells that naturally express the enzyme, or, alternatively, theenzyme can be obtained from cells transformed or transfected withexogenous DNA that encodes the DNA-PK enzyme. As another alternative,the enzyme can be purchased from commercial sources. In in vivo assays,cells that naturally express the DNA-PK enzyme are utilized.

[0122] Compounds that inhibit DNA-PK activity can be identified byincubating a test compound with a DNA-PK polypeptide and determining theeffect of the test compound on DNA-PK activity. The selectivity of acompound that inhibits the enzyme activity can be evaluated by comparingits effects on DNA-PK to its effect on other kinase enzymes.

[0123] Selective modulators include, for example, antibodies and otherproteins or peptides which specifically bind to a DNA-PK polypeptide,oligonucleotides which specifically bind to a DNA-PK polypeptide or aDNA-PK gene sequence, and other nonpeptide compounds (e.g., isolated orsynthetic organic and inorganic molecules) which specifically react witha DNA-PK polypeptide or a nucleic acid encoding the polypeptide.Presently preferred targets for the development of selective inhibitorsinclude, for example: (1) regions of the DNA-PK polypeptide that contactother proteins, (2) regions that localize the DNA-PK polypeptide withina cell wherein localization is required for specific kinase activity,(3) regions of the DNA-PK polypeptide that bind substrate, (4) regionsof the polypeptide that bind DNA and result in activation of kinaseactivity. Inhibitors of DNA-PK activity are therapeutically useful intreatment of a wide range of diseases and physiological conditions asdescribed herein.

[0124] Methods of the invention to identify inhibitors includevariations on any of the methods known in the art to identify bindingpartner compounds, the variations including techniques wherein a bindingpartner compound (e.g., a substrate molecule or a DNA sequence thatactivates the kinase) has been identified and a binding assay is carriedout in the presence and absence of a test inhibitor compound. Aninhibitor can be identified in those instances where the level ofbinding between the DNA-PK polypeptide and the binding partner compoundchanges in the presence of the test compound compared to the level ofbinding in the absence of the candidate modulator compound.

[0125] In addition to the assays described above, other methods arecontemplated which are designed to specifically identify DNA-PKinhibitors. In one aspect, methods of the invention use the split hybridassay, as generally described in WO98/13502. The invention also embracesvariations on this method, as described in WO95/20652.

[0126] The invention also contemplates high throughput screening (HTS)assays to identify compounds that inhibit DNA-PK biological activity(e.g., inhibit enzymatic activity, binding activity etc.). HTS assayspermit screening of large numbers of compounds in an efficient manner.Cell-based HTS systems are contemplated, including melanophore assays toinvestigate receptor-ligand interaction, yeast-based assay systems, andmammalian cell expression systems (Jayawickreme et al., Curr. Opin.Biotechnol. 8:629-634 (1997)). Automated and miniaturized HTS assays arealso embraced (Houston et al., Curr. Opin. BiotechnoL 8:734-740 (1997)).HTS assays are designed to identify “hits” or ‘lead compounds” havingthe desired property, from which modifications can be designed toimprove the desired property. Chemical modification of the “hit” or“lead compound” is often based on an identifiable structure/activityrelationship between the “hit” and the DNA-PK polypeptide.

[0127] There are a number of different libraries used for theidentification of compounds, and in particular small molecules, thatmodulate (i.e., increase or decrease) biological activity of apolypeptide of the invention, including, (1) organic and inorganicchemical libraries, (2) natural product libraries, and (3) combinatoriallibraries comprised of random peptides, oligonucleotides or organicmolecules.

[0128] Chemical libraries can be synthesized readily or purchased fromcommercial sources and consist of structural analogs of known compoundsor compounds that are identified as “hits” or “leads” via naturalproduct screening. The sources for natural product libraries arecollections from microorganisms (including bacteria and fungi), animals,plants and other vegetation, or marine organisms which are used tocreate mixtures for screening by: (1) fermentation and extraction ofbroths from soil, plant or marine microorganisms or (2) extraction ofplants or marine organisms. Natural product libraries includepolyketides, nonribosomal peptides, and variants (nonnaturallyoccurring) variants thereof. For a review, see Science 282:63-68 (1998).Combinatorial libraries are composed of large numbers of peptides,oligonucleotides, peptide nucleic acids, or organic compounds as amixture. They are relatively easy to prepare by traditional automatedsynthesis methods, PCR, cloning or proprietary synthetic methods. Ofparticular interest are peptide and oligonucleotide combinatoriallibraries. Still other libraries of interest include peptide, protein,peptidomimetic, multiparallel synthetic collection, recombinatorial, andpolypeptide libraries. For a review of combinatorial chemistry andlibraries created therefrom, see Myers, Curr. Opin. Biotechnol.8:701-707 (1997).

[0129] Identification of modulators through use of the various librariesdescribed herein permits modification of the candidate “hit” (or “lead”)to optimize the capacity of the “hit” to modulate activity. Compoundsthat are identified in the binding assays are then tested for antagonistor agonist activity in in vivo tissue culture or animal models that arewell known in the art.

[0130] The invention also provides methods of characterizing the potencyof a test compound as an inhibitor of a DNA-PK polypeptide, said methodcomprising the steps of: (a) measuring activity of a DNA-PK polypeptidein the presence of a test compound; (b) comparing the activity of theDNA-PK polypeptide in the presence of the test compound to the activityof the DNA-PK enzyme in the presence of an equivalent amount of areference compound of formula (I) or (II); wherein a lower activity ofthe DNA-PK polypeptide in the presence of the test compound than in thepresence of the reference compound indicates that the test compound is amore potent inhibitor than the reference compound, and a higher activityof the DNA-PK polypeptide in the presence of the test compound than inthe presence of the reference compound indicates that the test compoundis a less potent inhibitor than the reference compound.

[0131] The invention further provides methods of characterizing thepotency of a test compound as an inhibitor of a DNA-PK polypeptide, saidmethod comprising the steps of: (a) determining an amount of a controlcompound of formula (I) or (II) that inhibits an activity of a DNA-PKpolypeptide by a reference percentage of inhibition, thereby defining areference inhibitory amount for the control compound; (b) determining anamount of a test compound that inhibits an activity of a DNA-PKpolypeptide by a reference percentage of inhibition, thereby defining areference inhibitory amount for the test compound; (c) comparing thereference inhibitory amount for the test compound to a referenceinhibitory amount determined according to step (a) for the controlcompound of formula (I) or (II), wherein a lower reference inhibitoryamount for the test compound than for the control compound indicatesthat the test compound is a more potent inhibitor than the controlcompound, and a higher reference inhibitory amount for the test compoundthan for the control compound indicates that the test compound is a lesspotent inhibitor than the control compound. In one aspect, the methodutilizes a reference inhibitory amount which is the amount of thecompound that inhibits the activity of the DNA-PK polypeptide by 50%, by60%, by 70%, or by 80%. In another aspect, the method employs areference inhibitory amount that is the amount of the compound thatinhibits the activity of the DNA-PK polypeptide by 90%, by 95% or by99%. Methods of the invention can comprise determining the referenceinhibitory amount of the test compound in an in vitro biochemical assay,determining the reference inhibitory amount of the test compound in anin vitro cell-based assay, or determining the reference inhibitoryamount of the test compound in an in vivo assay.

[0132] Therapeutic Methods

[0133] The invention further provides methods of sensitizing a cell toan agent that induces a DNA lesion comprising the step of contacting thecell with one or more DNA-PK inhibitors of formula (I) or (II). Thepreferred compounds are set forth in Table 2. In presently preferredmethods, the agent that induces a DNA lesion is selected from the groupconsisting of radiation, exogenous chemicals, metabolite by-products,and combinations thereof. Particularly preferred methods include use ofone or more chemotherapeutic/anti-neoplastic agents as set out in Table3 that induce DNA lesions.

[0134] The invention further provides methods of potentiating atherapeutic regimen for treatment of cancer comprising the step ofadministering to an individual in need thereof an effective amount of aDNA-PK inhibitor of formula (I) or (II). Preferred compounds for use inthe methods are set out in Table 2. In one aspect, methods include thosewherein the therapeutic regimen for treatment of cancer is selected fromthe group consisting of chemotherapy, radiation therapy, and acombination of chemotherapy and radiation therapy. In methods whereinthe therapeutic regimen includes chemotherapy, the DNA-PK inhibitor isadministered before, concurrently with, and/or after administration ofthe chemotherapeutic/anti-neoplastic agent. In one aspect, methodsinclude use of one or more chemotherapeutic/anti-neoplastic agentsselected from the group consisting of those compounds set out in Table3. In another aspect of the invention, the DNA-PK inhibitor isadministered before, concurrently with, or after administration of acytokine, lymphokine, growth factor, or hematopoietic factor asdescribed herein.

[0135] Compounds of the invention are useful in instances whereradiation and chemotherapy are indicated to enhance the therapeuticbenefit of these treatments, including induction chemotherapy, primary(neoadjuvant) chemotherapy, and both adjuvant radiation therapy andadjuvant chemotherapy. In addition, radiation and chemotherapyfrequently are indicated as adjuvants to surgery in the treatment ofcancer. The goal of radiation and chemotherapy in the adjuvant settingis to reduce the risk of recurrence and enhance disease-free survivalwhen the primary tumor has been controlled. Chemotherapy is utilized asa treatment adjuvant for colon, lung, and breast cancer, frequently whenthe disease is metastatic. Adjuvant radiation therapy is indicated inseveral diseases including colon, lung, and breast cancers as describedabove. For example, radiation frequently is used both pre- andpost-surgery as components of the treatment strategy for rectalcarcinoma. Compounds of the invention therefore are particularly usefulfollowing surgery in the treatment of cancer in combination with radio-and/or chemotherapy.

[0136] The invention further relates to radiosensitizing tumor cellsutilizing a compound of formula (I) or (II). The preferred compounds arethose as described for the pharmaceutical compositions of the invention.Particular examples of the compounds suitable for the method include,but are not limited to, compounds as described in Table 2. A compoundthat can “radiosensitize” a cell, as used herein, is defined as amolecule, preferably a low molecular weight molecule, administered toanimals in therapeutically effective amount to increase the sensitivityof cells to electromagnetic radiation and/or to promote the treatment ofdiseases that are treatable with electromagnetic radiation. Diseasesthat are treatable with electromagnetic radiation include neoplasticdiseases, benign and malignant tumors, and cancerous cells.

[0137] Electromagnetic radiation treatment of other diseases not listedherein is also contemplated by the present invention. The terms“electromagnetic radiation” and “radiation” as used herein include, butare not limited to, radiation having the wavelength of 10⁻²⁰ to 1 meter.Preferred embodiments of the present invention employ theelectromagnetic radiation of: gammaradiation (10⁻²⁰ to 10⁻¹³ m), X-rayradiation (10⁻¹² to 10⁻⁹ m), ultraviolet light (10 nm to 400 mn),visible light (400 nm to 700 nm), infrared radiation (700 nm to 1.0 mm),and microwave radiation (1 mm to 30 cm).

[0138] Radiosensitizers are known to increase the sensitivity ofcancerous cells to the toxic effects of electromagnetic radiation.Several mechanisms for the mode of action of radiosensitizers have beensuggested. Hypoxic cell radiosensitizers (e.g., 2-nitroimidazolecompounds, and benzotriazine dioxide compounds) promote reoxygenation ofhypoxic tissue and/or catalyze generation of damaging oxygen radicals.Nonhypoxic cell radiosensitizers (e.g., halogenated pyrimidines) can beanalogs of DNA bases and preferentially incorporate into the DNA ofcancer cells and thereby promote the radiation ion-induced breaking ofDNA molecules and/or prevent the normal DNA repair mechanisms. Variousother potential mechanisms of action have been hypothesized forradiosensitizers in the treatment of disease.

[0139] Many cancer treatment protocols currently employ radiosensitizersactivated by electromagnetic radiation, e.g., X-rays. Examples ofX-ray-activated radiosensitizers include, but are not limited to, thefollowing: metronidazole, misonidazole, desmethylmisonidazole,pimonidazole etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233,E09, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR),5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine(FUdR), hydroxyurea, cisplatin, and therapeutically effective analogsand derivatives of the same.

[0140] Photodynamic therapy (PDT) of cancers employs visible light asthe radiation activator of the sensitizing agent. Examples ofphotodynamic radiosensitizers include the following, but are not limitedto: hematoporphyrin derivatives, PHOTOFRIN®, benzoporphyrin derivatives,NPe6, tin etioporphyrin (SnET2), pheoborbide-a, bacteriochlorophyll-a,naphthalocyanines, phthalocyanines, zinc phthalocyanine, andtherapeutically effective analogs and derivatives of the same.

[0141] Radiosensitizers can be administered in conjunction with atherapeutically effective amount of one or more other compounds,including but not limited to, compounds that promote the incorporationof radiosensitizers to the target cells; compounds that control the flowof therapeutics, nutrients, and/or oxygen to the target cells;chemotherapeutic agents that act on the tumor with or without additionalradiation; or other therapeutically effective compounds for treatingcancer or other disease. Examples of additional therapeutic agents thatcan be used in conjunction with radiosensitizers include, but are notlimited to, 5-fluorouracil (5-FU), leucovorin, oxygen, carbogen, redcell transfusions, perfluorocarbons (e.g., FLUOSOLW®-DA), 2,3-DPG,BW12C, calcium channel blockers, pentoxifylline, anti-angiogenesiscompounds, hydralazine, and L-BSO. Examples of chemotherapeutic agentsthat can be used in conjunction with radiosensitizers include, but arenot limited to, adriamycin, camptothecin, carboplatin, cisplatin,daunorubicin, doxorubicin, interferon (alpha, beta, gamma), interleukin2, irinotecan, docetaxel, paclitaxel, topotecan, and therapeuticallyeffective analogs and derivatives of the same.

[0142] The invention also can be practiced by including anotheranti-cancer chemotherapeutic agent with a compound of the invention,such as any conventional chemotherapeutic agent. The combination of theinhibitor compound with such other agents can potentiate thechemotherapeutic protocol. Numerous chemotherapeutic protocols known tothe skilled practitioner as being capable of incorporation into themethod of the invention. Any chemotherapeutic agent can be used,including alkylating agents, antimetabolites, hormones and antagonists,radioisotopes, as well as natural products. For example, the inhibitorcompound of the invention can be administered with antibiotics, such asdoxorubicin and other anthracycline analogs, nitrogen mustards, such ascyclophosphamide, pyrimidine analogs, such as 5-fluorouracil, cisplatin,hydroxyurea, taxol and its natural and synthetic derivatives, and thelike. As another example, in the case of mixed tumors, such asadenocarcinoma of the breast, where the tumors includegonadotropin-dependent and gonadotropin-independent cells, the compoundcan be administered in conjunction with leuprolide or goserelin(synthetic peptide analogs of LH-RH). Other anti-neoplastic protocolsinclude the use of an inhibitor compound with another treatmentmodality, e.g., surgery, radiation etc., also referred to herein as“adjunct anti-neoplastic modalities.” Thus, the method of the inventioncan be employed with such conventional regimens with the benefit ofreducing side effects and enhancing efficacy.

[0143] The present invention also provides methods of treating cancer inan animal, comprising administering to the animal an effective amount ofa compound that inhibits DNA-PK activity, such as a compound of formula(I) or (II). The preferred compound is selected from the group ofcompounds set forth in Table 2. The invention further is directed tomethods of inhibiting cancer cell growth, including processes ofcellular proliferation, invasiveness, and metastasis in biologicalsystems. Methods include use of a compound of formula (I) or (II) as aninhibitor of cancer cell growth. Preferably, the methods are employed toinhibit or reduce cancer cell growth, invasiveness, metastasis, or tumorincidence in living animals, such as mammals. Methods of the inventionalso are readily adaptable for use in assay systems, e.g., assayingcancer cell growth and properties thereof, as well as identifyingcompounds that affect cancer cell growth.

[0144] Compounds of the invention possess one or more desirable, butunexpected, combinations of properties, including increased activityand/or solubility, and reduction of adverse side effects. Thesecompounds have been found to inhibit cancer growth, includingproliferation, invasiveness, and metastasis, thereby rendering themparticularly desirable for the treatment of cancer. In particular,compounds of the invention exhibit cancer-inhibitory properties atconcentrations that appear to be substantially free of side effects.These compounds therefore are useful for extended treatment protocols,where the use of conventional chemotherapeutic compounds can exhibitundesirable side effects. For example, the co-administration of acompound of the invention with another, more toxic, chemotherapeuticagent can achieve beneficial inhibition of a cancer, while effectivelyreducing the toxic side effects in the patient.

[0145] In addition, the properties of hydrophilicity and hydrophobicityof the compounds of the invention are well balanced, thereby enhancingtheir utility for both in vitro and especially in vivo uses, while othercompounds lacking such balance are of substantially less utility.Specifically, compounds of the invention have an appropriate degree ofsolubility in aqueous media which permits absorption and bioavailabilityin the body, while also having a degree of solubility in lipids whichpermits the compounds to traverse the cell membranes, including thenuclear membrane, to a putative site of action. Thus, compounds of theinvention are maximally effective when delivered to the site of thetumor and they enter the tumor cells.

[0146] The cancers treatable by methods of the present inventionpreferably occur in mammals. Mammals include, for example, humans andother primates, as well as pet or companion animals, such as dogs andcats, laboratory animals, such as rats, mice and rabbits, and farmanimals, such as horses, pigs, sheep, and cattle.

[0147] Tumors or neoplasms include growths of tissue cells in which themultiplication of the cells is uncontrolled and progressive. Some suchgrowths are benign, but others are termed “malignant” and can lead todeath of the organism. Malignant neoplasms or “cancers” aredistinguished from benign growths in that, in addition to exhibitingaggressive cellular proliferation, they can invade surrounding tissuesand metastasize. Moreover, malignant neoplasms are characterized in thatthey show a greater loss of differentiation (greater“dedifferentiation”) and their organization relative to one another andtheir surrounding tissues. This property is also called “anaplasia.”

[0148] Neoplasms treatable by the present invention also include solidtumors, i.e., carcinomas and sarcomas. Carcinomas include thosemalignant neoplasms derived from epithelial cells which infiltrate(invade) the surrounding tissues and give rise to metastases.Adenocarcinomas are carcinomas derived from glandular tissue, or fromtissues which form recognizable glandular structures. Another broadcategory of cancers includes sarcomas, which are tumors whose cells areembedded in a fibrillar or homogeneous substance like embryonicconnective tissue. The invention also enables treatment of cancers ofthe myeloid or lymphoid systems, including leukemias, lymphomas, andother cancers that typically do not present as a tumor mass, but aredistributed in the vascular or lymphoreticular systems.

[0149] DNA-PK activity can be associated with various forms of cancerin, for example, adult and pediatric oncology, growth of solidtumors/malignancies, myxoid and round cell carcinoma, locally advancedtumors, metastatic cancer, human soft tissue sarcomas, including Ewing'ssarcoma, cancer metastases, including lymphatic metastases, squamouscell carcinoma, particularly of the head and neck, esophageal squamouscell carcinoma, oral carcinoma, blood cell malignancies, includingmultiple myeloma, leukemias, including acute lymphocytic leukemia, acutenonlymphocytic leukemia, chronic lymphocytic leukemia, chronicmyelocytic leukemia, and hairy cell leukemia, effusion lymphomas (bodycavity based lymphomas), thymic lymphoma lung cancer, including smallcell carcinoma, cutaneous T cell lymphoma, Hodgkin's lymphoma,non-Hodgkin's lymphoma, cancer of the adrenal cortex, ACTH-producingtumors, nonsmall cell cancers, breast cancer, including small cellcarcinoma and ductal carcinoma, gastrointestinal cancers, includingstomach cancer, colon cancer, colorectal cancer, polyps associated withcolorectal neoplasia, pancreatic cancer, liver cancer, urologicalcancers, including bladder cancer, including primary superficial bladdertumors, invasive transitional cell carcinoma of the bladder, andmuscle-invasive bladder cancer, prostate cancer, malignancies of thefemale genital tract, including ovarian carcinoma, primary peritonealepithelial neoplasms, cervical carcinoma, uterine endometrial cancers,vaginal cancer, cancer of the vulva, uterine cancer and solid tumors inthe ovarian follicle, malignancies of the male genital tract, includingtesticular cancer and penile cancer, kidney cancer, including renal cellcarcinoma, brain cancer, including intrinsic brain tumors,neuroblastoma, astrocytic brain tumors, gliomas, metastatic tumor cellinvasion in the central nervous system, bone cancers, including osteomasand osteosarcomas, skin cancers, including malignant melanoma, tumorprogression of human skin keratinocytes, squamous cell cancer, thyroidcancer, retinoblastoma, neuroblastoma, peritoneal effusion, malignantpleural effusion, mesothelioma, Wilms's tumors, gall bladder cancer,trophoblastic neoplasms, hemangiopericytoma, and Kaposi's sarcoma.Methods to potentiate treatment of these and other forms of cancer areembraced by the invention.

[0150] The invention is particularly illustrated herein in reference totreatment of certain types of experimentally defined cancers. In theseillustrative treatments, standard state-of-the-art in vitro and in vivomodels have been used. These methods can be used to identify agents thatcan be expected to be efficacious in in vivo treatment regimens.However, it will be understood that the method of the invention is notlimited to the treatment of these tumor types, but extends to any tumorderived from any organ system. Cancers whose invasiveness or metastasisis associated with DNA-PK expression or activity are especiallysusceptible to being inhibited or even induced to regress by means ofthe invention.

[0151] In addition to the neoplastic conditions described above, DNA-PKactivity can be correlated with other pathologies including aberrantapoptotic mechanisms, such as abnormal caspase activity; aberrant enzymeactivity associated with cell cycle progression, include for examplecyclins A, B, D and E; alterations in viral (e.g., Epstein-Barr virus,papillomavirus) replication in latently infected cells; chromosomestructure abnormalities, including genomic stability in general,unrepaired chromosome damage, telomere erosion (and telomeraseactivity), breakage syndromes including for example, Sjögren's syndrome,Bloom's syndrome, and Nijmegen breakage syndrome; embryonic stem celllethality; abnormal embryonic development; sensitivity to ionizingradiation; acute immune complex alveolitis; and Fanconi anemia.Treatment of these pathological conditions, and others that arise fromenhanced DNA-PK activity, also is embraced by the invention.

[0152] The present invention also includes methods to inhibit retroviralinfection utilizing a compound of the invention. DNA-PK participates innonhomologous end joining (NHEJ) of chromosomal DNA and retroviral DNAintegration into the host genome in accomplished through this type ofNHEJ reaction (Daniel et al., Science, 284:644-647 (1999)). Inhibitionof DNA-PK therefore can prevent retroviral DNA from integrating into thehost genome in infected cells. Because retroviral genomic integrationoccurs after infections, it is unlikely that inhibition of DNA-PKaffects early stages of infection. Instead, inhibiting DNA-PK preventsrepair of chromosomal breakage associated with integration and thereforesignal apoptosis for the infected cell. Assays to assess the ability ofDNA-PK inhibitors to act in this manner can be carried out by measuringapoptosis with virally infected cells in the presence and absence of aDNA-PK inhibitor.

[0153] Because many anti-cancer drugs are also immunosuppressive, theDNA-PK inhibitors also can be used to potentiate the efficacy of drugsin the treatment of inflammatory diseases. In particular, the method ofthe invention can be employed to treat humans therapeutically orprophylactically who are or may subject to an inflammatory disorder.“Inflammatory disorder” as used herein can refer to any disease,disorder, or syndrome in which an excessive or unregulated inflammatoryresponse leads to excessive inflammatory symptoms, host tissue damage,or loss of tissue finction. “Inflammatory disorders” can also refer topathological states mediated by influx of leukocytes and or neutrophilchemotaxis.

[0154] “Inflammation” as used herein refers to a localized, protectiveresponse elicited by injury or destruction of tissues, which serves todestroy, dilute, or wall off (sequester) both the injurious agent andthe injured tissue. Inflammation is notably associated with influx ofleukocytes and or neutrophil chemotaxis. Inflammation can result frominfection with pathogenic organisms and viruses and from noninfectiousmeans, such as trauma or reperfusion following myocardial infarction orstroke, immune response to foreign antigen, and autoimmune responses.Accordingly, inflammatory disorders amenable to the invention encompassdisorders associated with reactions of the specific defense system aswell as with reactions of the nonspecific defense system.

[0155] As used herein, the term “specific defense system” refers to thecomponent of the immune system that reacts to the presence of specificantigens. Examples of inflammation resulting from a response of thespecific defense system include the classical response to foreignantigens, autoimmune diseases, and delayed type hypersensitivityresponse mediated by T-cells. Chronic inflammatory diseases, therejection of solid transplanted tissue and organs, e.g., kidney and bonemarrow transplants, and graft versus host disease (GVHD) are fuirtherexamples of inflammatory reactions of the specific defense system.

[0156] The term “nonspecific defense system” as used herein refers toinflammatory disorders that are mediated by leukocytes that areincapable of immunological memory (e.g., granulocytes, macrophages).Examples of inflammation that result, at least in part, from a reactionof the nonspecific defense system include inflammation associated withconditions such as adult (acute) respiratory distress syndrome (ARDS) ormultiple organ injury syndromes; reperfusion injury; acuteglomerulonephritis; reactive arthritis; dermatoses with acuteinflammatory components; acute purulent meningitis or other centralnervous system inflammatory disorders such as stroke; thermal injury;inflammatory bowel disease; granulocyte transfusion associatedsyndromes; and cytokine-induced toxicity.

[0157] “Autoimmune disease” as used herein refers to any group ofdisorders in which tissue injury is associated with humoral orcell-mediated responses to the body's own constituents. “Allergicdisease” as used herein refers to any symptoms, tissue damage, or lossof tissue function resulting from allergy. “Arthritic disease” as usedherein refers to any disease that is characterized by inflammatorylesions of the joints attributable to a variety of etiologies.“Dermatitis” as used herein refers to any of a large family of diseasesof the skin that are characterized by inflammation of the skinattributable to a variety of etiologies. “Transplant rejection” as usedherein refers to any immune reaction directed against grafted tissue(including organs or cells, e.g., bone marrow), characterized by a lossof function of the grafted and surrounding tissues, pain, swelling,leukocytosis, and thrombocytopenia.

[0158] The therapeutic methods of the present invention include methodsfor the amelioration of disorders associated with inflammatory cellactivation. “Inflammatory cell activation” refers to the induction by astimulus (including, but not limited to, cytokines, antigens orauto-antibodies) of a proliferative cellular response, the production ofsoluble mediators (including but not limited to cytokines, oxygenradicals, enzymes, prostanoids, or vasoactive amines), or cell surfaceexpression of new or increased numbers of mediators (including, but notlimited to, major histocompatability antigens or cell adhesionmolecules) in inflammatory cells (including but not limited tomonocytes, macrophages, T lymphocytes, B lymphocytes, granulocytes(polymorphonuclear leukocytes including neutrophils, basophils, andeosinophils), mast cells, dendritic cells, Langerhans cells, andendothelial cells). It will be appreciated by persons skilled in the artthat the activation of one or a combination of these phenotypes in thesecells can contribute to the initiation, perpetuation, or exacerbation ofan inflammatory disorder.

[0159] The present invention enables methods of treating variousdiseases associated with or characterized by inflammation, for example,arthritic diseases such as rheumatoid arthritis, osteoarthritis, goutyarthritis, spondylitis; Behcet's disease; sepsis, septic shock,endotoxic shock, gram negative sepsis, gram positive sepsis, and toxicshock syndrome; multiple organ injury syndrome secondary to septicemia,trauma, or hemorrhage; ophthalmic disorders, such as allergicconjunctivitis, vernal conjunctivitis, uveitis, and thyroid-associatedophthalmopathy; eosinophilic granuloma; pulmonary or respiratorydisorders, such as asthma, chronic bronchitis, allergic rhinitis, ARDS,chronic pulmonary inflammatory disease (e.g., chronic obstructivepulmonary disease), silicosis, pulmonary sarcoidosis, pleurisy,alveolitis, vasculitis, pneumonia, bronchiectasis, and pulmonary oxygentoxicity; reperfusion injury of the myocardium, brain, or extremities;fibrosis, such as cystic fibrosis; keloid formation or scar tissueformation; atherosclerosis; autoimmune diseases such as systemic lupuserythematosus (SLE), autoimmune thyroiditis, multiple sclerosis, someforms of diabetes, and Reynaud's syndrome; transplant rejectiondisorders, such as GVHD and allograft rejection; chronicglomerulonephritis; inflammatory bowel diseases such as Crohn's disease,ulcerative colitis and necrotizing enterocolitis; inflammatorydermatoses, such as contact dermatitis, atopic dermatitis, psoriasis, orurticaria; fever and myalgias due to infection; central or peripheralnervous system inflammatory disorders, such as meningitis, encephalitis,and brain or spinal cord injury due to minor trauma; Sjogren's syndrome;diseases involving leukocyte diapedesis; alcoholic hepatitis; bacterialpneumonia; antigen-antibody complex mediated diseases; hypovolemicshock; Type I diabetes mellitus; acute and delayed hypersensitivity;disease states due to leukocyte dyscrasia and metastasis; thermalinjury; granulocyte transfusion associated syndromes; andcytokine-induced toxicity.

[0160] Methods of the invention can be used with animal models in orderto assess the efficacy of compounds of the invention. For example,animal models used in the study of inflammatory bowel disease (EBD) aregenerally elicited by intrarectal administration of noxious irritants(e.g., acetic acid or trinitrobenzene sulfonic acid/ethanol). Colonicinflammation induced by these agents is the result of chemical ormetabolic injury and lacks the chronic and spontaneously relapsinginflammation associated with human IBD. However, a recently describedmodel using subserosal injections of purifiedpeptidoglycan-polysaccharide (PG-PS) polymers from either group A orgroup D streptococci appears to be a more physiologically relevant modelfor human IBD (Yamada et al., Gastroenterology, 104:759-771 (1993)).

[0161] In this model, PG-PS is injected into the subserosal layer of thedistal colon. The resulting inflammatory response is biphasic with aninitial acute episode three days after injection, which is followed by aspontaneous chronic phase three to four weeks later. The late phaseresponse is granulomatous in nature, and results in colonic thickening,adhesions, colonic nodules and mucosal lesions. In general,granulomatous lesions are the result of chronic inflammation which leadsto the recruitment and subsequent activation of cells of themonocyte/macrophage lineage. In addition to mucosal injury, PG-PScolitis frequently leads to arthritis, anemia, and granulomatoushepatitis. The extraintestinal manifestations of the disease make themodel attractive for studying Crohn's colitis in that a significantnumber of patients with active Crohn's disease suffer from arthriticjoint disease and hepatobiliary inflammation.

[0162] Methods of the invention have particular utility in treatinghumans who are or may be subject to reperfusion injury, i.e., injuryresulting from situations in which a tissue or organ experiences aperiod of ischemia followed by reperfusion. The term “ischemia” refersto localized tissue anemia due to obstruction of the inflow of arterialblood. Transient ischemia followed by reperfusion characteristicallyresults in neutrophil activation and transmigration through theendothelium of the blood vessels in the affected area. Accumulation ofactivated neutrophils in turn results in generation of reactive oxygenmetabolites, which damage components of the involved tissue or organ.This phenomenon of “reperfusion injury” is commonly associated withconditions such as vascular stroke (including global and focalischemia), hemorrhagic shock, myocardial ischemia or infarction, organtransplantation, and cerebral vasospasm. To illustrate, reperfusioninjury occurs at the termination of cardiac bypass procedures or duringcardiac arrest when the heart, once prevented from receiving blood,begins to reperfuse. It is expected that inhibition of DNA-PK expressionor activity will result in reduced amounts of reperfusion injury in suchsituations.

[0163] With respect to the nervous system, global ischemia occurs whenblood flow to the entire brain ceases for a period. Global ischemia canresult from cardiac arrest. Focal ischemia occurs when a portion of thebrain is deprived of its normal blood supply. Focal ischemia can resultfrom thromboembolic occlusion of a cerebral vessel, traumatic headinjury, edema, or brain tumor. Even if transient, both global and focalischemia can cause widespread neuronal damage. Although nerve tissuedamage occurs over hours or even days following the onset of ischemia,some permanent nerve tissue damage may develop in the initial minutesfollowing the cessation of blood flow to the brain. Much of this damagehas been attributed to glutamate toxicity and to the secondaryconsequences of tissue reperfusion, such as the release of vasoactiveproducts by damaged endothelium and the release of cytotoxic products,such as free radicals and leukotrienes, by the damaged tissue.

[0164] Ischemia also can occur in the heart in myocardial infarction andother cardiovascular disorders in which the coronary arteries have beenobstructed as a result of atherosclerosis, thrombus, or spasm. Forexample, the method of the invention is believed to be useful fortreating cardiac tissue damage, particularly damage resulting fromcardiac ischemia or caused by reperfusion injury in mammals.

[0165] Administration

[0166] The compounds and pharmaceutical compositions of the inventioncan be administered to humans and other animals by any suitable route.For example, the compositions can be administered orally, includingsublingually, rectally, parenterally, intracisternally, intravaginally,intraperitoneally, topically and transdermally (as by powders,ointments, or drops), bucally, or nasally. The term “parenteral”administration as used herein refers to modes of administration otherthan through the gastrointestinal tract, which include intravenous,intramuscular, intraperitoneal, intrasternal, intramammary, intraocular,retrobulbar, intrapulmonary, intrathecal, subcutaneous andintraarticular injection and infusion. Surgical implantation also iscontemplated, including, for example, embedding a composition of theinvention under the splenic capsule, brain, or in the cornea.

[0167] Compounds of the present invention also can be administered inthe form of liposomes. As is known in the art, liposomes generally arederived from phospholipids or other lipid substances. Liposomes areformed by mono- or multi-lamellar hydrated liquid crystals that aredispersed in an aqueous medium. Any nontoxic, physiologicallyacceptable, and metabolizable lipid capable of forming liposomes can beused. The present compositions in liposome form can contain, in additionto a compound of the present invention, stabilizers, preservatives,excipients, and the like. The preferred lipids are the phospholipids andthe phosphatidyl cholines (lecithins), both natural and synthetic.Methods to form liposomes are known in the art. See, for example,Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, NewYork, N.Y. (1976), p. 33, et seq.

[0168] The compounds of the present invention can be used in the form ofpharmaceutically acceptable salts derived from inorganic or organicacids. By “pharmaceutically acceptable salt” is meant those salts whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of humans and lower animals without unduetoxicity, irritation, allergic response and the like, and arecommensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge etal., describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 66:1 (1977). The salts can be prepared in situduring the final isolation and purification of the compounds of theinvention or separately by reacting a free base fimction with a suitableacid. Representative acid addition salts include, but are not limited toacetate, adipate, alginate, citrate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, camphorate, camphorolsulfonate,digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate,fumarate hydrochloride, hydrobromide, hydrojodide,2-hydroxyethanesulfonate (isothionate), lactate, maleate,methanesulfonate, nicotinate, 2-Naphthalenesulfonate, oxalate, pamoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, phosphate, glutamate,bicarbonate, p-toluenesulfonate and undecanoate. Examples of acids thatcan be employed form pharmaceutically acceptable acid addition saltsinclude inorganic acids, such as hydrochloric acid, hydrobromic acid,sulfuric acid, and phosphoric acid, and organic acids, such as oxalicacid, maleic acid, succinic acid, and citric acid.

[0169] Basic nitrogen-containing groups can be quatemized with suchagents as lower alkyl halides such as methyl, ethyl, propyl, and butylchlorides, bromides, and iodides; dialkyl sulfates like dimethyl,diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl,lauryl, myristyl, and stearyl chlorides, bromides, and iodides; andarylalkyl halides, like benzyl and phenethyl bromides and others. Wateror oil-soluble or dispersible products thereby are obtained.

[0170] Basic addition salts can be prepared in situ during the finalisolation and purification of compounds of this invention by reacting acarboxylic acid-containing moiety with a suitable base, such as ahydroxide, carbonate, or bicarbonate of a pharmaceutically acceptablemetal cation or with ammonia or organic primary, secondary, or tertiaryamine. Pharmaceutically acceptable basic addition salts include, but arenot limited to, cations based on alkali metals or alkaline earth metals,such as lithium, sodium, potassium, calcium, magnesium, and aluminum,and the like, and nontoxic quaternary ammonia and amine cationsincluding ammonium, tetramethylammonium, tetraethyl-ammonium,methylamine, dimethylamine, trimethylamine ethylamine, diethylamine,triethylamine, and the like. Other representative organic amines usefulfor the formation of base addition salts include ethylenediamine,ethanolamine, diethanolamine, piperidine, piperazine, and the like.

[0171] Dosage forms for topical administration of a compound of thisinvention include powders, sprays, ointments, and inhalants as describedherein. The active compound is mixed under sterile conditions with apharmaceutically acceptable carrier and any needed preservatives,buffers, or propellants which may be required. Ophthalmic formulations,eye ointments, powders, and solutions also are contemplated as beingwithin the scope of this invention.

[0172] Parenteral Administration

[0173] Pharmaceutical compositions of the invention for parenteralinjection comprise pharmaceutically acceptable sterile aqueous ornonaqueous solutions, dispersions, suspensions, or emulsions, as well assterile powders for reconstitution into sterile injectable solutions ordispersions just prior to use. Examples of suitable aqueous andnonaqueous carriers, diluents, solvents, or vehicles include waterethanol, polyols (such as, glycerol, propylene glycol, polyethyleneglycol, and the like), and suitable mixtures thereof, vegetable oils(such, as olive oil), and injectable organic esters such as ethyloleate. Proper fluidity can be maintained, for example, by the use ofcoating materials such as lecithin, by the maintenance of the requiredparticle size in the case of dispersions, and by the use of surfactants.

[0174] These compositions also can contain adjuvants such aspreservatives, wetting agents, emulsifying agents, and dispersingagents. Prevention of the action of microorganisms can be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It also may bedesirable to include isotonic agents such as sugars, sodium chloride,and the like. Prolonged absorption of the injectable pharmaceutical formcan be brought about by the inclusion of agents which delay absorption,such as aluminum monostearate and gelatin.

[0175] In some cases, in order to prolong the effect of the drug, it isdesirable to slow the absorption of the drug from subcutaneous orintramuscular injection. This result can be accomplished by the use of aliquid suspension of crystalline or amorphous materials with poor watersolubility. The rate of absorption of the drug then depends upon itsrate of dissolution which, in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of a parenterallyadministered drug from is accomplished by dissolving or suspending thedrug in an oil vehicle.

[0176] Injectable depot forms are made by forming microencapsulematrices of the drug in biodegradable polymers such apolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations also are prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

[0177] The injectable formulations can be sterilized, for example, byfiltration through a bacterial- or viral-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved or dispersed in sterile water orother sterile injectable medium just prior to use.

[0178] Oral Administration

[0179] The invention provides methods for oral administration of apharmaceutical composition of the invention. Oral solid dosage forms aredescribed generally in Remington's Pharmaceutical Sciences, 18th Ed.,1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89. Solid dosageforms for oral administration include capsules, tablets, pills, powders,troches or lozenges, cachets, pellets, and granules. Also, liposomal orproteinoid encapsulation can be used to formulate the presentcompositions (as, for example, proteinoid microspheres reported in U.S.Pat. No. 4,925,673). Liposomal encapsulation may include liposomes thatare derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556).In general, the formulation includes a compound of the invention andinert ingredients which protect against degradation in the stomach andwhich permit release of the biologically active material in theintestine.

[0180] In such solid dosage forms, the active compound is mixed with, orchemically modified to include, a least one inert, pharmaceuticallyacceptable excipient or carrier. The excipient or carrier preferablypermits (a) inhibition of proteolysis, and (b) uptake into the bloodstream from the stomach or intestine. In a most preferred embodiment,the excipient or carrier increases uptake of the compound, overallstability of the compound and/or circulation time of the compound in thebody. Excipients and carriers include, for example, sodium citrate ordicalcium phosphate and/or (a) fillers or extenders such as starches,lactose, sucrose, glucose, cellulose, modified dextrans, mannitol, andsilicic acid, as well as inorganic salts such as calcium triphosphate,magnesium carbonate and sodium chloride, and commercially availablediluents such as FAST-FLO™, EMDEX™, STA-RX 1500™, EMCOMPRESS™ andAVICEL™.; (b) binders such as, for example, methylcelluloseethylcellulose, hydroxypropyhnethyl cellulose, carboxymethylcellulose,gums (e.g., alginates, acacia), gelatin, polyvinylpyrrolidone, andsucrose, (c) humectants, such as glycerol, (d) disintegrating agents,such as agar-agar, calcium carbonate, potato or tapioca starch, alginicacid, certain silicates, sodium carbonate, starch including thecommercial disintegrant based on starch, EXPLOTAB™, sodium starchglycolate, AMBERLITE™, sodium carboxymethylcellulose, ultramylopectin,gelatin, orange peel, carboxymethyl cellulose, natural sponge,bentonite, insoluble cationic exchange resins, and powdered gums such asagar, karaya or tragacanth; (e) solution retarding agents such aparaffm, (f) absorption accelerators, such as quaternary ammoniumcompounds and fatty acids including oleic acid, linoleic acid, andlinolenic acid (g) wetting agents, such as, for example, cetyl alcoholand glycerol monosterate, anionic detergent surfactants including sodiumlauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodiumsulfonate, cationic detergents, such as benzalkonium chloride orbenzethonium chloride, nonionic detergents including lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 40, 60, 65, and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose; (h)absorbents, such as kaolin and bentonite clay, (i) lubricants, such astalc, calcium sterate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, polytetrafluoroethylene (PTFE), liquid paraffin,vegetable oils, waxes, CARBOWAX™ 4000, CARBOWAX™ 6000, magnesium laurylsulfate, and mixtures thereof; (j) glidants that improve the flowproperties of the drug during formulation and aid rearrangement duringcompression that include starch, talc, pyrogenic silica, and hydratedsilicoaluminate. In the case of capsules, tablets, and pills, the dosageform also can comprise buffering agents.

[0181] Solid compositions of a similar type also can be employed asfillers in soft and hard-filled gelatin capsules, using such excipientsas lactose or milk sugar, as well as high molecular weight polyethyleneglycols and the like.

[0182] The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells, such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They optionally can contain opacifying agents and also can be of acomposition that they release the active ingredients(s) only, orpreferentially, in a part of the intestinal tract, optionally, in adelayed manner. Exemplary materials include polymers having pH sensitivesolubility, such as the materials available as EUDRAGIT®. Examples ofembedding compositions which can be used include polymeric substancesand waxes.

[0183] The active compounds also can be in micro-encapsulated form, ifappropriate, with one or more of the above-mentioned excipients.

[0184] Liquid dosage forms for oral administration includepharmaceutically acceptable emulsions, solutions, suspensions, syrups,and elixirs. In addition to the active compounds, the liquid dosageforms can contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol ethyl carbonate ethyl acetate,benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethyl formamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydroflirfurylalcohol, polyethylene glycols, fatty acid esters of sorbitan, andmixtures thereof.

[0185] Besides inert diluents, the oral compositions also can includeadjuvants, such as wetting agents, emulsifying and suspending agents,sweetening, coloring flavoring, and perfuming agents. Oral compositionscan be formulated and further contain an edible product, such as abeverage.

[0186] Suspensions, in addition to the active compounds, can containsuspending agents such as, for example ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, andmixtures thereof.

[0187] Pulmonary Administration

[0188] Also contemplated herein is pulmonary delivery of a DNA-PKinhibitor (or derivatives thereof). The inhibitor is delivered to thelungs of a mammal while inhaling, thereby promoting the traversal of thelung epithelial lining to the blood stream. See, Adjei et al.,Pharmaceutical Research 7:565-569 (1990); Adjei et al., InternationalJournal of Pharmaceutics 63:135-144 (1990) (leuprolide acetate); Braquetet al., Journal of Cardiovascular Pharmacology 13 (suppl.5): s.143-146(1989)(endothelin-1); Hubbard et al., Annals of Internal Medicine3:206-212 (1989)(ocl-antitrypsin); Smith et al., J. Clin. Invest.84:1145-1146 (1989) (α1-proteinase); Oswein et al., “Aerosolization ofProteins,” Proceedings of Symposium on Respiratory Drug Delivery II,Keystone, Colorado, March, 1990 (recombinant human growth hormone); Debset al., The Journal of Immunology 140:3482-3488 (1988) (interferon-γ andtumor necrosis factor α) and Platz et al., U.S. Pat. No. 5,284,656(granulocyte colony stimulating factor).

[0189] Contemplated for use in the practice of this invention are a widerange of mechanical devices designed for pulmonary delivery oftherapeutic products, including, but not limited to, nebulizers, metereddose inhalers, and powder inhalers, all of which are familiar to thoseskilled in the art.

[0190] Some specific examples of commercially available devices suitablefor the practice of the invention are the ULTRAVENT® nebulizer,manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the ACORN II®nebulizer, manufactured by Marquest Medical Products, Englewood, Colo.;the VENTOL® metered dose inhaler, manufactured by Glaxo Inc., ResearchTriangle Park, N.C.; and the SPINHALER® powder inhaler, manufactured byFisons Corp., Bedford, Mass.

[0191] All such devices require the use of formulations suitable for thedispensing of a compound of the invention. Typically, each formulationis specific to the type of device employed and can involve the use of anappropriate propellant material, in addition to diluents, adjuvants,and/or carriers useful in therapy.

[0192] The inhibitor composition is prepared in particulate form,preferably with an average particle size of less than 10 μm, and mostpreferably 0.5 to 5 μm, for most effective delivery to the distal lung.

[0193] Carriers include carbohydrates such as trehalose, mannitol,xylitol, sucrose, lactose, and sorbitol. Other ingredients for use informulations may include lipids, such as DPPC, DOPE, DSPC and DOPC,natural or synthetic surfactants, polyethylene glycol (even apart fromits use in derivatizing the inhibitor itself), dextrans, such ascyclodextran, bile salts, and other related enhancers, cellulose andcellulose derivatives, and amino acids.

[0194] Also, the use of liposomes, microcapsules or microspheres,inclusion complexes, or other types of carriers is contemplated.

[0195] Formulations suitable for use with a nebulizer, either jet orultrasonic, typically comprise a compound of the invention dissolved inwater at a concentration of about 0.1 to 25 mg of biologically activeprotein per mL of solution. The formulation also can include a bufferand a simple sugar (e.g., for protein stabilization and regulation ofosmotic pressure). The nebulizer formulation also can contain asurfactant to reduce or prevent surface-induced aggregation of theinhibitor composition caused by atomization of the solution in formingthe aerosol.

[0196] Formulations for use with a metered-dose inhaler device generallycomprise a finely divided powder containing the inhibitor compoundsuspended in a propellant with the aid of a surfactant. The propellantcan be any conventional material employed for this purpose, such as achlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid also can be useful as a surfactant.

[0197] Formulations for dispensing from a powder inhaler device comprisea finely divided dry powder containing the inhibitor and also caninclude a bulking agent, such as lactose, sorbitol, sucrose, mannitol,trehalose, or xylitol, in amounts which facilitate dispersal of thepowder from the device, e.g., 50 to 90% by weight of the formulation.

[0198] Other Routes of Administration

[0199] Nasal delivery of the inhibitor also is contemplated. Nasaldelivery allows the passage of the protein to the blood stream directlyafter administering the therapeutic product to the nose, without thenecessity for deposition of the product in the lung. Formulations fornasal delivery include those with dextran or cyclodextran. Delivery viatransport across other mucous membranes also is contemplated.

[0200] Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of theinvention with suitable nonirritating excipients or carriers, such ascocoa butter, polyethylene glycol, or suppository wax, which are solidat room temperature, but liquid at body temperature, and therefore meltin the rectum or vaginal cavity and release the active compound.

[0201] In order to facilitate delivery of compounds across cell and/ornuclear membranes, compositions of relatively high hybrophobicity arepreferred. Compounds can be modified in a manner which increaseshydrophobicity, or the compounds can be encapsulated in hydrophobiccarriers or solutions which result in increased hydrophobicity.

[0202] Dosages

[0203] Actual dosage levels of active ingredients in the pharmaceuticalcompositions of the invention can be varied to obtain an amount of theactive compound(s) that is effective to achieve the desired therapeuticresponse for a particular patient, compositions, and mode ofadministration. The selected dosage level depends upon the activity ofthe particular compound, the route of administration, the severity ofthe condition being treated, and the condition and prior medical historyof the patient being treated. However, it is within the skill of the artto start doses of the compound at levels lower than required to achievethe desired therapeutic effort and to gradually increase the dosageuntil the desired effect is achieved.

[0204] Generally dosage levels of about 0.1 to about 1000 mg, about 0.5to about 500 mg, about 1 to about 250 mg, about 1.5 to about 100, andpreferably of about 5 to about 20 mg of active compound per kilogram ofbody weight per day are administered orally or intravenously. Ifdesired, the effective daily dose can be divided into multiple doses forpurposes of administration, e.g., two to four separate doses per day.

[0205] The invention is exemplified by the following examples. Example 1describes DNA-PK enzyme purification. Example 2 sets forth the standardDNA-PK enzyme assay. Example 3 describes a high throughput assay used toidentify inhibitors of DNA-PK. Example 4 addresses determination ofselectivity of the DNA-PK inhibitors identified. Example 5 relates toassessing cellular toxicity of the DNA-PK inhibitors. Example 6describes a V(D)J recombination assay. Example 7 provides an assay todetermine the chemosensitization capacity of DNA-PK inhibitors. Example8 addresses the ability of DNA-PK inhibitors to affect long term cellproliferation. Example 9 relates to assessment of the ability of arepresentative DNA-PK inhibitor to potentiate the toxic effect ofradiation treatment. Example 10 describes the ability of DNA-PKinhibitors to reduce/inhibitor tumor formation in animal models. Example11 addresses use of DNA-PK inhibitors in the treatment of humandiseases. Examples 12-149 provide a description of methods for synthesisand physical properties of exemplary DNA-PK inhibitors of the invention.

EXAMPLE 1 DNA-PK Enzyme Purification

[0206] In order to develop an assay to screen for enzyme inhibitors, amethod for large scale purification of human DNA-PK was performed(Lees-Miller et al., Mol. Cell. Biol. 10:6472-6481 (1990)).

[0207] HeLa S3 cells (ATCC CCL-2.2; Batch F12594) were raised inMEM-Joklik media (Gibco) supplemented with 10% FBS, 100 U/mL penicillinand 100 μg/mL streptomycin at 37° C. in a humidified chamber under 5%CO₂. For enzyme purification, cells were grown to a density ofapproximately 1×10⁶ cells/mL in two spinner flasks each containing 6 Lmedia. Cells were collected by centrifugation for 10 min at 1,000 rpm ina GS-6R Beckman centrifuge. Cell pellets were washed 1× in ice cold PBSand collected by centrifugation. Cells were resuspended in ice cold LSBbuffer, containing 10 mM HEPES-KOH, pH 7.2, 25 mM KCl, 10 mM NaCl, 1 mMMgCl₂, 0.1 mM EDTA, and 0.1 mM DTT, and collected by centrifugation for10 min at 2,000 rpm. Cell pellets were resuspended in an equal volume ofbuffer, allowed to stand on ice for 5 min, and then frozen in liquidnitrogen.

[0208] The frozen HeLa cell pellet was thawed at 37° C. and immediatelycentrifuged at 10,000×g in a Beckman JA10 rotor for 20 min at 4° C. Theresulting supernatant (S10 faction, fraction 1) was collected, solidPMSF was added to 0.5 mM final concentration, and the resulting mixturewas centrifuged at 100,000×g in a Beckman Type 45Ti rotor for 3 h at 4°C. The pelleted material was resuspended in H buffer (containing 25 mMHEPES-KOH, pH 7.5, 0.2 mM EDTA, 0.5 mM DTT, 0.5 M KCl, and 10 mM MgCl₂)and centrifuged at 100,000×g for 1 h at 4° C. H Buffer was added to thesupernatant until the ionic strength was equal to that of H buffercontaining 0.1 M KCl (S100-2 fraction, also fraction II).

[0209] The S100-2 fraction was applied to a 28 mL Q-SEPHAROSE® FF column(1.5×16 cm) equilibrated in H buffer with 0.1 M KCl. The resin waswashed with 5 column volumes and developed with a 140 mL linear gradient(0.1 to 0.5 M KCl in H buffer) at a flow rate of 1.5 mL/min. A broadpeak of activity was eluted, pooled, and dialyzed into H buffer with 0.1M KCl (fraction III).

[0210] Fraction III was applied to an 8 mL SP-SEPHAROSE® FF column (1×10cm) equilibrated in H buffer with 0.1 M KCl. The resin was washed with 5column volumes and developed with a 30 mL linear gradient (0.1 to 0.5 MKCl in H buffer) at a flow rate of 1.5 mL/min. Active fractions werepooled (fraction IV) and stored at −70° C.

EXAMPLE 2 Standard DNA-PK Assay

[0211] Standard kinase reactions used to measure phosphorylation of ap53 peptide substrate (SEQ ID NO: 1) contained, in 20 μL, 25 mMHEPES-KOH, pH 7.5, 10 mM MgCl₂, 0.5 mM DTT, 50 μM ATP, 0.01 mCi/mL[γ-³²P]ATP, 10 μg/mL replicative form m (RFM) DNA, 200 μM p53 peptideand 0.2 μg purified DNA-PK (as described in Example 1).

[0212] Glu-Pro-Pro-Leu-Ser-Gln-Glu-Ala-Phe-Ala-Asp-Leu-Trp-Lys-Lys-ArgSEQ ID NO: 1

[0213] Reactions were carried out at room temperature. Reactions werestarted by addition of ATP and stopped by application tophosphocellulose paper. Reaction products spotted onto phosphocellulosepaper were washed five times with a total volume of at least 250 mL 10%acetic acid or 150 mM phosphoric acid. The paper was air dried andradioactivity was determined in a Beckman LS6000IC scintillationcounter.

[0214] The DNA-PK kinase activity was found to be stimulated 17-fold inthe presence of linear duplex RFIII DNA and activity was dependent uponaddition of polypeptide substrate. The K_(m) and V_(max) for ATPconsumption were found to be 6.6 μM ATP and 1.2 pmol ATP/min,respectively. Enzyme activity was inhibited by wortmannin with anIC₅₀=100 to 250 nM and by demethoxyviridin at an IC₅₀=5 nM.

EXAMPLE 3 High-Throughput DNA-PK Kinase Assay

[0215] In order to screen large numbers of compounds for the ability toinhibit DNA-PK activity, an automated high throughput screening assaywas developed. The steps in the automated procedure are as set outbelow.

[0216] A stock [³²P]-ATP mixture was prepared containing 0.5 mM p53peptide (SEQ ID NO: 1), 125 μM ATP, 25 μg/mL DNA, 2.5×DNA-PK Buffer(62.5 HEPES-KOH, pH 7.5, 25 mM MgCl₂, 1.25 mM DTT), and 6.25 μCi/mL³²P-ATP. The stock mixture was prepared in a 100 mL volume containing 25mL 2 mM p53 peptide, 2.5 μL 5 mM ATP, 2.5 mg DNA (volume depends onconcentration of DNA available), 50 mL 5×DNA-PK Buffer, 625 μL[³²P]-ATP, and 22.0 mL H₂O (minus volume of DNA used). Approximately 50mL of the mixture was dispensed into a reagent reservoir in the assayapparatus, and the remaining ³²P-ATP mix was stored at 4° C.

[0217] Enzyme dilution buffer (EDB) (containing 25 mM HEPES-KOH, pH 7.5,10 mM MgCl₂, 0.5 mM DTT) was diluted 1:2 (20 mL EDB diluted with 20 mLH₂O) and the diluted buffer was transferred to the assay apparatus. A1.0 mL aliquot of DNA-PK enzyme was thawed, 225 μL enzyme was added to45 mL diluted EDB, and the mixture was vortexed. The diluted enzyme wastransferred to the assay apparatus for the first round of assays, andthe remaining undiluted enzyme was dispensed into 150 μL aliquots. Forsubsequent sets of 15 plates, the 150 μL aliquots of DNA-PK enzyme wereadded to 30 mL of diluted EDB.

[0218] In the assay, a Sagian/SAMI protocol runs a program designated“DNA-PK” which included the following steps. In a first step, 100 μL of150 mM phosphoric acid was added to each well on a MILLIP ORE® filterplate. The phosphoric acid was vacuumed through and an assay plate withcontrols, a chemical plate, and a dilution plate containing 198 μL H₂Owere loaded. The program then added 2 μL assay chemical to dilutionplate, mixed the dilution plate, and transferred 20 μL of the dilutedchemical to the assay plate. The pipette tips were washed and 20 μL ofenzyme was transferred to the assay plate. The tips were washed againand 20 μL ³²P-ATP mix was added to the assay plate. The reaction wasmixed and incubated for 20 min.

[0219] The program then pipetted 10 μL 150 mM phosphoric acid into theassay plate and mixed the phosphoric acid with the assay mix to stop thereaction. The filter plates were allowed to incubate at least 10 minsbefore washing to ensure complete binding of the phosphorylated peptide.

[0220] After the 10 min incubation, the MILLIPORE® plate was transferredto a wash station and aspirated. Another 200 μL 1.5 mM phosphoric acidwas added to each well and the plate was aspirated again. This step wasrepeated four more times so that a total of five 200 μL phosphoric acidwashes were completed. Using an 8 channel pipette, 100 μL 95% ethanolwas added to each well and the liquid aspirated through. The plates wereblotted on paper towels several times and allowed to air dryapproximately 10 to 15 min. Scintillation fluid (50 μL) was added toeach well and radioactivity was counted using a Wallac beta counter.

[0221] Inhibitors of DNA-PK-catalyzed protein phosphorylation wereidentified through an analysis of an internal chemical library. Thisscreen yielded 47 compounds with IC₅₀ values less than 52 μM. Thesecompounds were defined as hits and tested further.

EXAMPLE 4 Selectivity Determination

[0222] The most potent inhibitors of DNA-PK identified in the highthroughput screening assay were tested for the ability to inhibitphosphorylation catalyzed by other kinase enzymes. In order todistinguish the DNA-PK specific inhibitors from general protein kinaseinhibitors, the inhibitors identified in Example 2 were used in assayswith distantly related (from a phylogenic standpoint) protein kinases(Hunter and Plowman, Trends Biochem Sci 22:18-22 (1997)) casein kinaseI, protein kinase Cθ and the calcium/calmodulin dependent kinase II. Toidentify which inhibitors preferentially bound to DNA-PK from a set ofmore closely related kinases, the compounds were assayed for inhibitoryactivity against the ataxia-telangiectasia related (ATR) protein kinase,the FK506-rapamycin associated protein kinase, and thephosphatidylinositol-3 kinase p110δ. Compounds that selectivelyinhibited phosphorylation catalyzed by DNA-PK were defined as specificinhibitors of DNA-PK. Selectivity of inhibition can be defined asfollows:

(IC₅₀(test enzyme))/(IC₅₀(DNA-PK))>50

[0223] All assays were carried out at room temperature in polypropylenemicrofuge tubes or polystyrene microtiter plates.

[0224] P110 Assay #1

[0225] DNA encoding epitope-tagged p110 δ (Chantry et al., J. Biol.Chem. 272:19236-19241 (1997)) was transfected into COS cells using DEAEdextran. Three days after transfection, the cells were serum-starvedovernight in Dulbecco's modified Eagle's medium plus 0.1% fetal bovineserum. The culture plates were rinsed once with calcium- andmagnesium-free phosphate-buffered saline and lysed in 3 mL of buffer R(containing 1% TRITON® X-100, 150 mM NaCl, 10 mM Tris, pH 7.4, 1 mMEDTA, 0.5% NONIDET® P-40, 0.2 mM phenylmethylsulfonyl fluoride, and 1%aprotinin) per confluent 150 mm dish. The p110δ enzyme wasimmunoprecipitated using an anti-FLAG monoclonal antibody M2 accordingto the manufacturer's suggested protocol. The immunoprecipitates werewashed three times with buffer R, twice with PAN buffer (containing 10MM PIPES, pH 7.0, 100 mM NaCl, and 20 μg/mL aprotinin), and resuspendedin PAN buffer. Standard kinase reactions used to measure phosphorylationof phosphatidylinositol contained, in 20 μL, 20 mM HEPES-KOH, pH 7.5, 5mM MnCl₂, 0.45 mM EGTA, 10 μM ATP, 0.2 mCi/mL [γ-³²P]ATP, 0.2 mg/mLphosphatidylinositol, immunoprecipitated p110δ, and variable amounts ofkinase inhibitor. Reactions were started by addition of ATP and stoppedby placing aliquots in 100 μL 1M HCl.

[0226] Reaction products were extracted with 200 μL CHCl₃:MeOH (1:1).The organic phase was extracted with 80 μL 1 M HCl/MeOH (1:1),subsequently dried, resuspended in 8 μL ice cold CHCl₃/MeOH (1:1) with0.1% HCl, and applied to a 1% potassium oxalate-impregnated silica 60thin layer chromatography plate. Reaction products were resolved byascending chromatography in CHCl₃/MeOH/NH₄OH (9:7:2) and visualized byautoradiography. Crude phospholipid standards were run in parallel withthe radiolabeled samples and visualized by exposing the plate to iodinevapor. Reactions were quantified by densitometry of autoradiograms usingPDQUEST with a pdi 325oe densitometer.

[0227] P110 Assay #2

[0228] Kinase reactions contained, in 30 μL, 20 mM HEPES-KOH, pH 7.5, 5mM MnCl₂, 0.45 mM EGTA, 32 μM ATP, 0.1 mCi/mL [γ-³²P]ATP, 0.2 mg/mLphosphatidylinositol, and purified p 110δ enzyme (described above), andvariable amounts of kinase inhibitor molecules. Reactions were startedby addition of ATP and stopped by placing aliquots in 100 μL 1N HCl.Reaction products were extracted with 100 μL CHCl₃ and phosphorylationof phosphatidylinositol was determined by measurement of radioactivityin the organic phase using a Beckman LS6000IC scintillation counter.

[0229] FK506-Rapamycin Associated Protein Kinase (FRAP) Assay

[0230] Kinase reactions contained, in 60 μL, 15 mM HEPES-KOH, pH 7.4, 10mM MnCl₂, 80 mM NaCl, 0.3 mg/mL BSA, 2 μM ATP, 0.02 mCi/mL [γ-³²P]ATP,0.2 mg/mL pH-acid stable protein (PHAS) substrate, purified recombinantFRAP kinase (Brown et al., Nature, 369:756-758 (1994)), and variableamounts of kinase inhibitor molecules. Reactions were started byaddition of ATP and terminated by adding aliquots to 20 μL 0.9 Mphosphoric acid. Reaction cocktails were transferred to P81 paper andwashed 5× with at least 250 mL 150 mM phosphoric acid. Phosphorylationof PHAS was determined by measurement of radioactivity bound to P81paper using a Beckman LS6000IC scintillation counter.

[0231] Ataxia Telangiectasia Related (ATR) Protein Kinase Assay

[0232] ATR from mouse testes was assayed by antibody capture. Microtiterplates coated with 200 ng mAb 224C (Plug et al., J. Cell Sci.111:413-423 (1998)) were incubated with 12.5 μg mouse testes extract andincubated at 4° C. for at least 18 h. Plates were washed with kinasebuffer (containing 25 mM HEPES-KOH, pH 7.5, 50 mM KCl, 10 mM MgCl₂, 2%glycerol, and 1 mM DTT) and incubated with 450 μM myoD peptide (DH-22),10 μM ATP, and 0.12 mCi/mL [γ-³²P]ATP in kinase buffer. Reactions (20μL) were stopped by addition of 180 μL 150 mM phosphoric acid andaliquots spotted onto P81 paper and washed 5× with at least 2 mL 150 mMphosphoric acid. Scintillation cocktail was added to the dried P81 paperand radioactivity was measured using a Wallac scintillation counter.

[0233] Protein Kinase Cθ (PKCθ) Assay

[0234] Standard reactions contained, in 30 μL, 20 mM Tris-HCl, pH 7.5,20 mM MgCl₂, 1 mM CaCl₂, PMA, PS, 0.3% TRITON® X-100, 70 μM ATP, 0.1mCi/mL [γ-³²P]ATP, 60 μM myelin basic protein (MBP) peptide (residues 4through 14), purified recombinant PKCO (Baier et al., J. Biol.Chem.268:4997-5004 (1993)) and variable amounts of kinase inhibitor.Reactions were started by addition of ATP and terminated by adding 5%phosphoric acid. Reactions were transferred to P81 paper and the paperwas washed 5× with at least 250 mL 150 mM phosphoric acid.Phosphorylation of MBP was determined by measurement of radioactivitybound to P81 paper using a Beckman LS6000IC.

[0235] Casein Kinase I (CKI) Assay

[0236] Standard 20 μL reactions contained 150 mM NaCl, 50 mM Tris-HCl,pH 7.5, 10 mM MgCl₂, 100 μM ATP, 0.1 mCi/mL [γ-³²P]ATP, 2.8 μg casein,and 100 ng purified recombinant CKI (Knippschild, Oncogene 15:1727-1736(1997)). Reactions were started by addition of ATP and stopped byapplication to P81 paper. Reaction products spotted onto P81 paper werewashed five times with a total volume of at least 250 mL 150 mMphosphoric acid, dried, and radioactivity determined in a BeckmanLS6000IC scintillation counter.

[0237] Calcium/Calmodulin Kinase II (CaMKII) Assay

[0238] Standard 20 μL reactions contained 50 mM HEPES-KOH, pH 7.5, 5 MMMgCl₂, 1 mM CaCl₂, 30 μg/mL calmodulin, 200 μM CaMKII peptide substrate(Calbiochem, La Jolla, Calif.), 50 AM ATP, 1 mCi/mL [γ-³²P]ATP, CaMKII(Calbiochem, La Jolla, Calif.) and variable amounts of kinaseinhibitors. Reactions were started by addition of ATP and terminated byadding aliquots to 10% acetic acid. Reactions were transferred to P81paper and washed 5× with greater than 250 mL 10% acetic acid.Phosphorylation of CaMKII peptide was determined by measurement ofradioactivity bound to P81 paper using a Beckman LS6000IC scintillationcounter.

EXAMPLE 5 Cellular Toxicity Determination

[0239] In order to assess toxicity of the DNA-PK inhibitors identified,each inhibitor was first incubated with cells in culture and cellviability monitored.

[0240] A large panel of cultured human tumor cell lines (see Table 4)ACHN, 786-0, HCT116, SW620, SK-MEL5, SK-MEL28, A549, H322, OVCAR3,SK-OV3, MCF7, MDA-MB231, MOLT4, HL60, SNB19, PC3) and the normal humanfibroblast cell line MRC5 were treated with2-hydroxy-4-morpholin-4-yl-benzaldehyde and1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone at concentrations up to200 μM. TABLE 4 CULTURED HUMAN CELL LINES ASSAYED CELL LINE CELL TYPEATCC DESIGNATION ACHN Human renal adenocarcinoma CRL-1611 786-O Humanrenal cell adenocarcinoma CRL-1932 HCT 116 Human colorectal carcinomaCCL-247 SW620 Human colorectal adenocarcinoma CCL-227 SK-MEL-5 Humanmalignant melanoma HTB-70 SK-MEL-28 Human malignant melanoma HTB-72 A549Human lung carcinoma CCL-185 NCI-H322 Human nonsmall cell lung carcinomaCRL-5806 OVCAR-3 Human ovary carcinoma HTB-161 SK-OV-3 Human ovarycarcinoma HTB-77 MCF-7 Human mammary gland adenocarcinoma HTB-22MDA-MB-231 Human mammary gland adenocarcinoma HTB-26 MOLT-4 Human acutelymphoblastic leukemia CRL-1582 HL-60 Human acute promyelocytic leukemiaCCL-240 SNB-19 Central nervous system PC-3 Human prostate adenocarcinomaCRL-1435

[0241] Cultured cells were distributed on 96-well microtiter plates andallowed to adhere for at least 16 h. Cells were treated with 200 μM(2-hydroxy-4-morpholin-4-yl-benzaldehyde or1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone continuously for 48 h.Cells were then analyzed using the MTS assay, CELLTITER™ cellproliferation assay kit (Promega, Madison, Wis.) according to themanufacturer's recommended protocol.

[0242] Using the MTS dye metabolism assay, no evidence of killing of anyof these cell lines was observed.

[0243] Cell viability was also tested during drug treatment using thetrypan blue exclusion assay. Freshly cultured thymocytes from BALB/c andBALB/c p53-/p53- mice were incubated in RPMI 1640 media (Gibco)containing 20% FBS, 1 mM sodium pyruvate , 2 mM L-glutamine, 100 U/mLpenicillin and 100 μg/mL streptomycin at 37° C. with 5% CO₂ in ahumidified incubator. At the time of preparation, p53+/p53+andp53/p53thyrnocytes exhibited 96% and 89% viability, respectively,measured by vital dye staining (trypan blue). Cultured cells wereincubated in the presence of varying concentrations of2-hydroxy-4-morpholin-4-yl-benzaldehyde up to 32 μM. Cultured human andmouse cell lines were incubated in RPMI 1640 media containing 10% FBS,100 U/mL penicillin and 100 μg/mL streptomycin at 37° C. with 5% CO₂ ina humidified incubator with 2-hydroxy-4-morpholin-4-yl-benzaldehyde forseveral days. Tumor cell lines, HL60, Jurkat, JY and B16/F10 and thenormal cell line WS1 were tested using this assay when incubated at drugconcentrations up to 25 μM.

[0244] In no instances were cells sensitive to killing by2-hydroxy-4-morpholin-4-yl-benzaldehyde as determined by the trypan blueexclusion method.

[0245] The lack of chemical cytotoxicity of these DNA-PK inhibitors wasconsistent with the high viability of cells cultured from scid mutantmice and showed no indication that essential cellular functions arenonspecifically targeted by either2-hydroxy-4-morpholin-4-yl-benzaldehyde or1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone.

EXAMPLE 6

[0246] (a) V(D)J Recombination Assay

[0247] Because scid mutant mice are unable to perform V(D)Jrecombination, assays were designed to assess whether the class ofDNA-PK inhibitors could also disrupt the process in vitro.

[0248] Recombination assays were performed as previously described (Leuet al., Mol. Cell. Biol 15:5657-5670 (1995)). DR3 cells were grown to adensity of approximately 0.5 to 0.8×10⁵ cells/mL in RPMI 1640 containing10% dialyzed FBS, L-glutamine, 100 U/mL penicillin and 100 μg/mLstreptomycin, 50 μM β-mercaptoethanol, and 10 μM methotrexate at 37° C.with 5% CO₂ in a humidified incubator. Cells were concentrated andwashed in HBS (containing 30 mM HEPES-KOH, pH 7.5, 140 mM NaCl, and 5 mMKCl) and resuspended in HBS at a density of 2.0×10⁸ cells/mL. Cells(1.0×10⁷) were transiently transfected by electroporation with 10 μg ofthe recombination substrate pJH200 (Leu et al., Mol. Cell. Biol.15:5657-5670 (1995)) and transferred to a 90 mm cell culture dishcontaining 8 mL media with or without2-hydroxy-4-morpholin-4-yl-benzaldehyde as described above. After 2days, cells were removed from the dish with a rubber policeman,concentrated, washed one time with PBS, and resuspended in 0.1 mL lysisbuffer (0.3% Triton X-100, 50 mM Tris-HCl, pH 7.5, 50 mM EDTA).Following a five min incubation at room temperature, cellular debris wasremoved by centrifugation at 12,000 rpm for two min in a microfuge. Thesupernatant was extracted sequentially with phenol andchloroform/isoamyl acetate (24:1), after which, one tenth volume 3 Msodium acetate and 2 volumes cold 100% ethanol were added to the aqueousphase. Precipitated nucleic acid was collected by centrifugation at12,000 rpm for 15 min and suspended in 25 μL water. An aliquot of thematerial was used to transform DH5α cells by electroporation. Cells wereplated on LB media containing either carbenicillin (carb) orcarbenicillin and chloramphenicol (cm). Recombination was defined as theratio of colonies resistant to both carbenicilin and chloramphenicol(carb^(r)cm^(r)) to colonies resistant to carbenicilin (carb^(r)).

[0249] Results indicated that V(D)J recombination was inhibited whencultured mouse B-lymphocytes, competent to rearrange immunoglobulinrecombination signal-coding sequences present on autonomouslyreplicating plasmids, were treated with2-hydroxy-4-morpholin-4-yl-benzaldehyde DNA rearrangement was inhibited8-fold at 50 μM 2-hydroxy-4-morpholin-4-yl-benzaldehyde indicating thatDNA-PK participates in V(D)J recombination in vitro and that this drugis capable of inhibiting intracellular DNA-PK.

[0250] (b) DNA Double-strand Break Repair Assay

[0251] To further determine the cellular effect of DNA-PK inhibitors, anassay to measure chromosomal discontinuities was employed. Ionizingradiation induces chromosomal DNA double-strand breaks. Following highdose radiation, chromosomes can be extracted from cells and fractionatedby pulse field electrophoresis to distinguish chromosomal fragments fromintact larger chromosomes. Using this technique, the activity of DNA-PKinhibitors was measured.

[0252] MDA-MB231 (human breast carcinoma) cells were seeded onto T25flasks with RPMI1640+10% FBS, 2 mM L-glutamine, penicillin G 100U/ml-streptomycin sulfate 10μg/ml, 1 mM Na pyruvate. When confluent,media was removed and replaced with media containing DNA-PK inhibitor orvehicle. Cells were incubated at 37° C. for 1 hr in a humidified chamberwith 5% CO₂. Media then was removed and flasks were filled with ice-coldD-PBS and either: (a) processed immediately; (b) irradiated (25 Gy in a¹³⁷Cs Mark I irradiator at a flux of 335 rad/min) and processedimmediately; (c) irradiated and incubated for 2 hr in completeRPM11640+vehicle at 37° C. (in humidified 5% CO₂ atmosphere to allow forDNA repair), or (d) irradiated and incubated for 2 hr in completeRPMIl640+DNA-PK inhibitor compound. To process cells, D-PBS or media wasreplaced with 5 ml ice-cold D-PBS and cells were removed from flasks,concentrated with cell resuspension buffer (10 mM Tris pH 7.2, 50 mMEDTA) and added to warm 2% clean cut agarose (Bio-Rad # 170-3594). Cellslurries were embedded in agarose, then incubated in PK buffer (10 nMTris pH 8.0, 100 MM EDTA, 1% lauryl sarcosine, 0.2% Na deoxycolate, 100μg/ml Proteinase K (Bio-Rad #732-6348)) at 4° C. for 2 min, followed byincubation at 50° C. overnight. Cells embedded in agarose plugs werewashed with buffer containing 10 mM Tris pH 8.0, 50 mM EDTA for 15 min.Agarose plugs then were treated with 50 μg/ml RNase (DNase free;Boehringer#1579-681) at 37° C. for 1 hr. An agarose gel (1% low meltagarose; Bio-Rad #162-0017) then was cast around plugs in 0.5×TBE andchromosomal DNA was fractionated by pulse field gel electrophoresis at99V (2V/cm), 45sec pulse time, 48hr with 14° C. recirculating 0.5×TBE ina CHEF-DR II cell apparatus (Bio-Rad). Chromosomal DNA was visualizedwith SYBR-Gold (Molecular probes # S-11494) and the fluorescent imagequantified on the STORM 860 (Molecular Dynamics).

[0253] Using this technique, it was found that concentrations of1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone which enhanced radiationinduced cell killing (measured by the DNA synthesis assay) alsoinhibited DNA double-strand break repair. These data demonstrate thatDNA-PK inhibitors perturb chromosomal DNA double-strand break repair,and suggests that inhibition of this DNA repair reaction is responsiblefor the potentiation of radiation toxicity. Furthermore, these datasuggest that DNA-PK inhibitors bind the target in the nucleus ultimatelyinducing sensitivity to chemical and physical agents that yield DNAdsbs.

[0254] (c) Animal Tumor Model: Pro-drug

[0255] To increase the solubility and achieve higher plasma drugconcentrations, the activity of a phoshorylated derivative of1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone, i.e., Example 34-p, wastested. Mouse pharmacokinetic analysis of Example 34-p demonstrated thatit is rapidly converted to parent compound (Example 34) in plasma.Example 34-p then was tested for anti-tumor activity.

[0256] HCT116 cells (human colon carcinoma) or MDA-MB-231 (human breastcarcinoma) were used to propagate xenograft tumors in 6-8 week oldfemale athymic BALB/c (nu/nu) mice. Mice were maintained in a laminarairflow cabinet under pathogen-free conditions and fed sterile food andwater ad libitum. Human cells were grown to subconfluence in RMP11640supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/mL streptomycinand 1.5 mM L-glumtamine in a 5% humidified environment. Single cellsuspensions were prepared in CMF-PBS and cell concentrations adjusted to4×10⁷ cells/mL. Mice were inoculated subcutaneously on the rightproximal leg with 4×10⁶ cells.

[0257] Mice were randomized (10 mice/group) into three groups with meantumor volumes ca. 100 mg. Mice then were either mock treated (drugvehicle only), irradiated only (150 rad), or received both ionizingradiation and drug (150 rad+32 mg Example 34-p). Example 34-p wasprepared in 25% CREMOPHORO: 75% normal saline at a concentration of 80mg/ml. Dosing was modeled to obtain plasma levels of >9,000 ng/ml forapproximately 5 hr. Animals were dosed with 100 [il of drug solution(400 mg/kg) and irradiated on the tumor bearing leg using a Mark I ¹³⁷Ceirradiator approximately 20 minutes after the initial bolus drugadministration. Animals subsequently were dosed every hour for a totalof 4 hrs. This dosing regimen, 4×400 mg/kg with one dose of 150 rad perday was repeated once (HCT116 tumors) or twice (MDA-MB231) for a totalof two or three treatment days, respectively. Animals were weighed andtumors were measured every other day for the duration of the experiment.Mice were sacrificed when the tumor volume reached 1200 mg orapproximately 7% of body weight.

[0258] Animals treated with Example 34-p and radiation showed asignificant (p=0.0414; HCT116 experiment) delay in tumor growth ratecompared to animals treated with vehicle or radiation only. The timerequired for the drug and radiation treated animals to reach 50%survival was about 2 weeks longer than for the study group receivingradiation only. These data demonstrate the utility of making anddelivering pro-drugs of this class of DNA-PK inhibitor compounds. Thesedata also show that Example 34-p has anti-tumor activity in twodifferent human (colon, breast)-mouse xenograft assays and may begenerally applicable to a wide range of human tumor types and cancerdisease indications.

EXAMPLE 7 Chemosensitization

[0259] To test the hypothesis that inhibition of DNA-PK can potentiatethe killing effect of cellular treatment that induces DNA dsbs, cellswere incubated in the presence of both selective DNA-PK inhibitors andchemical DNA damaging agents.

[0260] Cells plated at a density of 5,000 to 20,000 per well in 96-wellmicrotiter plates were grown in RMPI 1640 containing 10% FBS, 100 U/mLpenicillin and 100 μg/mL streptomycin, for 18 h at 37° C. in ahumidified incubator with 5% CO₂. Cells tested included ACHN, 786-0,HCT-116, SW620, SK-MEL-5, SK-MEL-28, A549, H322, OVCAR-3, SK-OV-3,MCF-7, MDA-MB-231, MOLT4, HL60, SNB-19, PC-3. Cells were treated withmedia containing chemotherapeutic drugs alone or with media containingchemotherapeutic drugs and a DNA-PK inhibitor of the invention,1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone. Cells were incubated fortwo days before growth was measured using a tetrazolium dye assay (MTS).Chemotherapeutic drugs included bleomycin etoposide, vinblastine,doxorubicin, paclitaxel, cisplatin, chlorambucil, cyclophosphamide,5-fluorouracil, cytosine-arabinoside, 6-mercaptoguanine and methotrexate(all purchased from Sigma). The drug concentration necessary to inhibitcell growth to 50% of untreated control cells was defined as the GI₅₀.

[0261] Results indicated that1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone enhanced growthinhibition of the human colon carcinoma cell line HCT116 when incubatedwith bleomycin etoposide and chlorambucil (agents known to induce DNAdsbs). The GI₅₀ (bleomycin) was reduced 7-fold in the presence of 100 μM1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone from 2×10⁻¹ units/mL to3×10⁻² units/mL. The etoposide GI₅₀ was reduced 10-fold from 1.2×10⁻⁴ Mto 1.4×10⁻⁵ M. The chlorambucil GI₅₀ was also reduced 10-fold from1.2×10⁻⁴ M to 1.4×10⁻⁵ M. The chorambucil GI₅₀ was also reduced 10-foldfrom 1.2×10⁻⁴M to 1.4×10⁻⁵M. Therefore,1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone potentiated the growthinhibitory effect of these three DNA damaging agents.

[0262] The inhibitor 1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone didnot potentiate the growth inhibitory effects of chemotherapeutic agentsthat do not induce DNA dsbs, such as vinblastine, doxorubicin,paclitaxel, cisplatin, cyclophosphamide, 5-fluorouracil,cytosine-arabinoside, 6-mercaptoguanine and methotrexate.

EXAMPLE 8 Chemo/Radiosensitization: Colony Forming Assay

[0263] In order to assess the ability of the DNA-PK inhibitors topotentiate the effect of radiation treatment on cancer cells, thefollowing assay was carried out.

[0264] Colon carcinoma cells (HCT 116) plated in RPMI-1640 containing10% dialyzed FBS, L-glutamine, 100 U/mL penicillin and 100 μg/mLstreptomycin with or without 200 μM2-hydroxy-4-morpholin-4-yl-benzaldehyde or1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone were treated withincreasing amounts of y-radiation up to 1600 rad. Alternatively, cellswere incubated with or without1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone and increasing amount ofetoposide up to 100 μM for 24 h, at which time the drug was removed andcells were incubated in fresh media without drug. After approximatelyone week, colonies were scored. Culture media was removed and cells werefixed with a solution of methanol/acetic acid (3:1 v/v). Followingfixation, PBS was added and colonies were visualized by staining withcrystal violet (50 μg/mL final concentration). Colonies were defined ascellular masses 25 to 40 cells in diameter and were estimated to be madeup of 500 to 1500 cells.

[0265] In addition to the 48 h growth assay, the effect of DNA-PKinhibitors on long term cellular growth was tested following treatmentthat induced DNA double-strand breaks. Continued cell division ofadherent cells in culture results in the formation of a colony. Colonyformation is the balance of growth and cell division with cell death byapoptosis, necrosis, or senescence. Because the clonogenic survivalassay measures a cell's ability to sustain growth and cell division, itprovides a more accurate representation of the effect of drug treatmenton cellular proliferation.

[0266] Using the clonogenic assay, it was observed that both2-hydroxy-4-morpholin-4-yl-benzaldehyde and1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone increased the sensitivityof the HCT116 cell line to killing by y-radiation. The radiation dosagewhich kills 50% of the cells (LD₅₀) for cells incubated in the presenceof 1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone was 4-fold lower thanwhen incubated in the absence of drug, i.e., 35 rad and 150 rad,respectively. Drug alone had no effect on cell division. Likewise, aradiation dose that kills 90% of the cells (LD₉₀) also was 4-fold lowerwhen cells were treated with drug than without, i.e., 100 rad and 410rad, respectively.

[0267] An alternative metric by which to characterize the cellularsensitivity to radiation is the slope, Do, of the survival curve duringthe exponential phase of cell killing (Radiobiology for the Radiologist,Fourth Edition, Hall (ed), J.B. Lippincott Co, Philadelphia, 1994; pp.37-38). More precisely, this measurement indicates the radiation doserequired to reduce cell survival e⁻¹. By this method,1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone reduced D₀ by one third,from 150 rad to 50 rad. These data clearly demonstrated that this classof DNA-PK inhibitors radiosensitive tumor cells.

[0268] These results were in agreement with experiments performed withscid cell lines and leukocytes cultured from wild-type and scid mice.Others (Fulop et al., Nature 347:479-482 (1990)) have demonstrated thatscid cells are 2.4-fold more radiosensitive than normal cells asmeasured by granulocyte-macrophage colony forming units (CFU-GM).Similarly, others (Hendrickson et al., Proc. Natl. Acad. Sci. USA.,88:4061-4065 (1991)) have observed that cultured scid fibroblasts were2.9-fold more sensitive than normal mouse fibroblasts. In light of theseobservations, these data strongly suggest that these compoundsradiosensitized tumor cells by disrupting DNA dsbs repair as a result ofsubcellular inhibition of DNA-PK.

[0269] To further confirm that inhibition of DNA dsbs repair by thisclass of DNA-PK enzyme inhibitors enhances tumor cell killing, assayswere designed to determine if the inhibitor compounds could sensitizecells to killing by etoposide, a chemical agent that induces DNA dsbs.In experiments set up essentially as described above, it was observedthat, as with radiation, the cytotoxicity of etoposide was potentiatedby 1-(2-hydroxy-4-morpholin-4-yl-phenyl-ethanone. The LD₅₀ of etoposidewith and without 1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone was0.075 μM and 1.1 μM, respectively; a 15-fold enhancement. The LD₉. wasenhanced 3-fold by 1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone, 0.9FM versus 2.9 μM. This class of DNA-PK inhibitors therefore enhancedcytotoxicity of DNA double-strand breaks induced by both physical andchemical DNA damaging agents.

EXAMPLE 9 Radiosensitization: DNA Synthesis Assay

[0270] In order to assess the ability of a representative DNA-PKinhibitor to potentiate the toxic effect of radiation treatment on alarge number of cancer cells, the following assay was performed.

[0271] Cell lines represented in Table 4 were used to inoculate 96 wellmicrotitre plates at a density of 5×10³ cells/mL in RPMI 1640 containing10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. Cells wereincubated with or without 1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanoneat concentrations ranging from 15-125 μM and treated with γ-radiation atdoses up to 800 rads. Cells were incubated in a humidified chamber at37° C. in 5% CO₂ for five days. After four days, cells were pulsed with(³H]-thymidine. On day five, cells were harvested and cellularradioactivity determined.

[0272] All cell lines exhibited a y-radiation dose-dependent decrease inthe absolute amount of [³H]-thymidine incorporated into cellular DNA.[³H]-thymidine incorporation was further reduced by incubation with the1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone DNA-PK inhibitor in adose-dependent manner, with the drug-dependent decrease in DNA synthesisinduced by γ-radiation ranging from 1.8- to 4-fold. The effect of1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone (as a representative ofthis class of DNA-PK inhibitors) enhanced the toxic effect ofγ-rradiation on all tumor cells described in Table 4.

[0273] This data corroborated the previous observations in the colonyforming assay (Example 8), showing that inhibition of DNA-PK sensitizestumor cells to treatments that induce cellular DNA double-strand breakdamage. Furthermore, these results indicated that the DNA-PK inhibitorsof the invention are applicable to treatment of a wide range of tumorsfrom major organ systems.

EXAMPLE 10 Animal Tumor Models

[0274] In order to determine if the results obtained above could beextended to in vivo conditions, an animal model was designed using humantumor cell xenografts.

[0275] HCT116 cells (human colon carcinoma) were used to propagatexenograft tumors in 6-8 week old female athymic BALB/c (nu/nu) mice.Mice were maintained in a laminar airflow cabinet under pathogen-freeconditions and fed sterile food and water ad libitum. HCT116 cells weregrown to subconfluence in McCoy's media supplemented with 10% FBS, 100U/mL penicillin, 100 μg/mL streptomycin, and 1.5 mM L-glutamine in a 5%CO₂ humidified environment. Single cell suspensions were prepared inCMF-PBS (0.9 mM KCl, 2.7 mM KH₂PO₄, 13.8 mM NaCl, pH 7.5), and cellconcentration adjusted to 4×10⁷ cells/mL. Mice were inoculatedsubcutaneously (s.c.) on the right flank or right leg with a total of4×10⁶ cells (100 μL).

[0276] Mice were randomized (5 mice/group) into four treatment groupsand used when tumors reached a weight of 75 to 100 mg (usually 7 to 11days post-inoculation). Tumors were measured with vernier calipers andtumor weights were estimated using the empirically-derived formulabelow.

[0277] Tumor weight (mg)=tumor length (mm)×tumor width (mm)2/3.3.

[0278] Treatment consisted of i) 100 μL s.c. injection of vehicle alone(1:3, CREMOPHOR® (BASF) EL:D-PBS, 0.9 mM CaCl₂, 0.5 mM MgCl₂, 0.9 mMKCl, 2.7 mM KH₂PO₄, 13.8 mM NaCl.); ii) 100 μL 8 mg/mL2-hydroxy-4-morpholin-4-yl-benzaldehyde in vehicle (approximately 40mg/kg); iii) vehicle plus 2.5 Gy γ-irradiation 30 min post-injection; oriv) 100 μL 8 mg/mL 2-hydroxy-4-morpholin-4-yl-benzaldehyde in vehicle(approximately 40 mg/kg) plus 2.5 Gy γ-radiation 30 min later. Allinjections were performed s.c. in the vicinity of the tumor. Radiationwas delivered with a using a ¹³⁷Ce source. Treatments were repeateddaily for 1 to 5 consecutive days. Tumor size was monitored every otherday for the duration of the experiment.

[0279] Based on previous results, it was expected that tumor cellkilling would be most effective if DNA repair and signaling weredisrupted at the time of DNA damage induction. Pharmacokinetic analysisof two DNA-PK inhibitors, 2-hydroxy-4-morpholin-4-yl-benzaldehyde and1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone, indicated that themaximum plasma concentration for each was achieved at approximately 15to 30 min after administration and remained high for about one hourfollowing subcutaneous administration. The protocol therefore wasdesigned whereby mice were irradiated within 40 min following drugadministration so that plasma drug concentration would be near itszenith during radiation treatment.

[0280] Preliminary experiments indicated that2-hydroxy-4-morpholin-4-yl-benzaldehyde enhanced the tumoristatic effectof total body irradiation. In experiments where animals were given100-500 rad γ-radiation, 2-hydroxy-4-morpholin-4-yl-benzaldehyde delayedtumor growth 1.2- to 1.8-fold relative to animals that receivedradiation only. When larger doses of radiation were delivered,2-hydroxy-4-morpholin-4-yl-benzaldehyde treatment had a tumoricidaleffect. In an experiment where animals with an average tumor burden of500 mg were treated with drug and 300 rad once daily for two days,tumors shrank greater than 10-fold. In contrast, irradiated animalsreceiving the same radiation treatment without drug demonstrated nodecrease in tumor size although the rate of tumor regrowth was retarded.

[0281] Treatment with 1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanonealso potentiated the effect of radiation on tumors. Mice receiving 250rad plus drug once daily for five consecutive days exhibited no increasein tumor growth whereas tumors in mice receiving radiation aloneincreased at a rate of 7 mg per day.

[0282] These data demonstrated that the DNA-PK inhibitors potentiate thekilling effect of radiation on tumors by increasing theirradioresponsiveness. The inhibitors, therefore, act synergistically withradiation.

EXAMPLE 11 Use of DNA-PK Inhibitors in the Treatment of Human Disease

[0283] The observations described herein indicated that the DNA-PKinhibitors have broad applications in the treatment of proliferativedisorders, including cancer. In particular, the inhibitors potentiatethe therapeutic effects of radiation during the treatment of humancancers and can be used in combination with chemotherapy and severalforms of radiation treatments including, teletherapy (i.e., radiationtherapy administered from a source at a distance from the body),radioimmunotherapy, and brachytherapy (i.e., radiation therapy whereinthe irradiation source is close to or within the body). The radiationcan be administered by stereotactic radiosurgery or fractionatedmicrobeam teletherapy.

[0284] During teletherapy, drug administration prior to radiationtreatment, as was carried out in the animal experiments described above,is most effective in reducing tumor mass. In this method, the drug isadministered systemically and radiation is focused locally to the tumorsite. Circumstances also can exist wherein it is advantageous toadminister the DNA-PK inhibitor following radiation treatment. In eithertreatment, the drug can be delivered by any of a number of routesdescribed herein. Potentiation of the efficacy of teletherapy can beapplied to radiocurative tumors as well as radiorefractory tumors.Seminoma, a carcinoma of the cervix, larynx, breast and prostate,Hodgkin's disease, and acute lymphocytic leukemia are examples ofradiocurative tumors for which this class of DNA-PK inhibitors improvetreatment by achieving greater therapeutic effect and reducingcollateral tissue toxicity. Combination therapy using the drug withteletherapy also has the effect of enhancing the radioresponsiveness ofradioresistant tumors; some examples include as glioblastomas,osteogenic sarcomas, retinoblastomas, astrocytomas, and some head andneck cancers. It is anticipated that inhibition of DNA-PK activity canbe of therapeutic benefit in all instances where radiation is used withcurative intent.

[0285] Radiation therapy also is indicated for pain management duringcancer treatment. Palliation of pain is an important component of sometreatment strategies. It is contemplated that the procedure of radiationwith palliative intent also is enhanced by inhibition of DNA-PK in tumorand possible normal tissue, e.g., administration of bone-localizingisotopes, such as Sn-117, for the treatment of bone pain associated withbone cancer.

[0286] The DNA-PK inhibitors of the invention also are effective incombination with radioimmunotherapy and brachytherapy. The goal in thesetherapies is to deliver radiation internally to tumor sites in anattempt to minimize damage to surrounding normal tissue, radioactiveseed implants for prostate cancer. The DNA-PK inhibitors can be used toenhance the therapeutic index of these radiation treatments also.

[0287] DNA-PK inhibitors of the invention also can be used to potentiatethe benefits of chemotherapy. Combination treatment withchemotherapeutic agents that induce DNA damage and a DNA-PK inhibitorinduces a synergistic effect on tumor tissue as observed in experimentsusing etoposide, bleomycin, and chlorambucil with cultured human tumorcells. These data indicate that treatment regimens employingtopoisomerase inhibitors, alkylating agents, and/or bleomycin areenhanced by this class of DNA-PK inhibitor. Other chemical agents usedin the treatment of cancer can also be made more effective by inhibitionof DNA-PK.

[0288] Therapeutic benefit also can be obtained through theadministration of a DNA-PK inhibitor conjugated to an antibody. Drugdelivery can be targeted to specific sites within the body as a functionof the determinants of antibody recognition. This method ofadministration can be combined with radiation or chemotherapy. It isexpected that DNA-PK inhibitor drugs can be coadministered withchemotherapeutic drugs which themselves are linked to tumor-specificantibodies.

[0289] It also is expected that DNA-PK inhibitors can be used incombination with nongenotoxic modulators of the cell division cycle withor without genotoxic treatments, such as radiation and chemotherapydescribed above. Such nongenotoxic treatments are anticipated to perturbcell cycle metabolism, affecting the temporal order and kinetics of cellcycle events such as initiation of the cell cycle, DNA replication,centrosome duplication, chromosome segregation and cytokinesis. Theexecution of these cell cycle events is integrated with events relatedto DNA damage repair. Therefore, the combined effect of disrupting thecoordinated repair of DNA damage with cell cycle progression is expectedto reduce the fidelity of the cell division cycle with lethalconsequences.

[0290] Because many anti-cancer drugs are also immunosuppressive, theDNA-PK inhibitors also can be used to potentiate the efficacy of drugsin the treatment of inflammatory diseases. Examples of some diseasesthat can benefit from combination therapy with the inhibitors arerheumatoid arthritis, psoriasis, vitiligo, Wegener's granulomatosis, andsystemic lupus erythematosus (SLE). A common theme in the treatment ofarthritis, Wegener's granulomatosis, and SLE is the use ofimmunosuppressive therapies such as ionizing radiation, methotrexate,and cyclophosphamide. As these treatments induce, DNA damage, eitherdirectly or indirectly, inhibition of DNA-PK activity within offendingimmune cells will render the cells more sensitive to control by thesestandard treatments. Psoriasis and vitiligo are commonly treated withultraviolet radiation (UV) in combination with psoralens. These two DNAdamaging agents induce T cell killing thought to be responsible for thisdisease. Ihibition of DNA-PK enhances the killing effect of UV radiationand psoralens, and increases the therapeutic index of the treatmentregimen. In general, the DNA-PK inhibitors can potentiate the control ofinflammatory disease cells in combination with currently usedimmunosuppressive drugs.

[0291] Recently, it has been demonstrated that cells cultured from scidmice are refractory to retrovirus infection (Daniel et al., Science,284: 644-647 (1999)) due to the deficiency in DNA-PK. This class ofDNA-PK inhibitors therefore can be used to protect cells from retroviralinfection. These inhibitors can have therapeutic benefit in thetreatment of acquired immune deficiency syndrome (AIDS) by blocking HIVinfection of T-cells. In this example, this class of inhibitors can havesignificant activity as a single agent or coadministered with otherantiviral agents, such as protease inhibitors; transcriptase inhibitors,nucleoside analogs, and the like.

[0292] To the degree that DNA-PK participates in retroviral infection,inhibitors of the invention can be used in therapeutic intervention. TheRNA genome of retroviruses is copied into DNA which integrates into thegenome of an infected cell. Integration necessarily requiresintroduction of dsbs in the host cell genome, and observations suggest arole for DNA-PK is repairing the break (Daniel et al., Science,284:644-647 (1999)). Inhibition of DNA-PK therefore arrests cell growthand signal apoptosis of the infected cell.

[0293] Preliminary results using retrovirus-infected Jurkat J77 cellsindicated that apoptosis increased 1.5- to 2-fold in cells treated witha DNA-PK inhibitor compared to cells that were not treated.

[0294] The inhibitors of the invention also can be effective duringmarrow ablation prior to bone marrow transplantation. Bone marrowconditioning is currently performed by treatment with cytotoxic agentssuch as ionizing radiation, cyclophosphamide, and/or busulfan. The goalof the procedure is to remove existing marrow cells and provide spacefor transplanted stem cells to take residence. The inhibitors thereforecan potentiate the cytotoxic effect of current treatments by allowingmore effective bone marrow conditioning with less toxic side effects.

EXAMPLES 12-149 Description of DNA-PK inhibitors

[0295] Compounds structurally related to the DNA-PK-specific inhibitorsmentioned above also were tested for kinase inhibitory activity asdescribed above. Synthesis and physical properties of these inhibitorsare set out below.

EXAMPLE 12 5-Morpholin-4-yl-2-nitro-phenylamine

[0296] A solution of 5-chloro-2-nitrophenylamine (4.3 g, 25 mmol) andmorpholine (4.4 mL, 500 mmol) in dimethylsulfoxide (DMSO) (25 mL) wasstirred at 90° C. for 16 h. The reaction mixture was poured into water(500 mL) and the resulting precipitate was collected by vacuumfiltration and recrystallized from methanol (300 mL) to yield theproduct as orange crystals (2.4 g, 43%). M.P. 177-178° C. (lit. 184-186°C.). See, Polymers 36:3401-3408 (1995).

[0297]¹H NNIR (90 MHz, CDCl₃) δ8.02 (d, J=9.2 Hz, 1H), 6.32-6.09 (m,4H), 3.82 (t, J=5.0 Hz, 4H), 3.29 (t, J=5.0 Hz, 4H). LRMS: 223 (M⁺).

EXAMPLE 13 5-(4-Methyl-piperazin-1-yl)-2-nitro-phenylamine

[0298] A mixture of 5-chloro-2-nitrophenylamine (2.5 g, 14 mmol),1-methylpiperizine (3.2 mL, 30 mmol), and potassium carbonate (K₂CO₃)(2.0 g, 14 mmol) in dimethylformamide (DMF) (20 mL) was stirred at 120°C. for 18 h. The reaction mixture was cooled to room temperature andpoured into water (150 mL). The precipitated crude product was collectedby filtration, dried in vacuo, and purified by recrystallization fromethyl acetate (EtOAc) (75 mL) to yield yellow-green crystals (1.8 g,54%). See, J. Med. Chem., 39:997 (1996).

[0299]¹H NMR (90 MHz, CDCl₃) δ8.00 (d, J =9.9 Hz, 1H), 6.33-6.15 (m,3H), 5.94 (d, J=2.6 Hz, 1H), 3.37 (t, J=5.3 Hz, 4H), 2.51 (t, J=5.3 Hz,4H), 2.33 (s, 3H).

EXAMPLE 14 2-Hydroxymethyl-5-morpholin-4-yl-phenol

[0300] A solution of sodium borohydride (NaBH₄) (0.66 g, 17 mmol) inwater (25 mL) was treated with 4-morpholinylsalicylaldehyde (1.8 g, 9mmol) in small portions over 30 min and stirred at room temperature for3 h. The reaction was quenched with acetone (5.1 mL, 100 mmol),acidified to pH 7 with 7.8 mL of 5% hydrochloric acid (aqueous) andextracted with chloroform (CHCl₃) (2×100 mL). The combined CHCl₃extracts were dried over sodium sulfate (Na₂SO₄) and concentrated invacuo to yield 0.7 g of pale yellow solid which was purified bytrituration with 60/40 CHCl₃/ethyl acetate (EtOAc) to yield the productas a white solid (0.25 g).

[0301]¹H NMR (90 MHz, CDCl₃) δ6.90 (d, J=8.0 Hz, 1H), 6.43-6.34 (m, 2H),4.77 (s, 3H), 3.82 (t, J=4.6 Hz, 4H), 3.09 (t, J=4.6 Hz, 4H).

EXAMPLE 15 2-Nitro-5-thiomorpholin-4-yl-phenylamine

[0302] A mixture of 5-chloro-2-nitrophenylamine (2.5 g, 14 mmol),thiomorpholine (3.0 g, 30 mmol), and K₂CO₃ (2.0 g, 14 mmol) in DMF (20mL) was stirred at 120° C. for 18 h. The reaction mixture was cooled toroom temperature and poured into water (150 mL). The crude product thatprecipitated was collected by filtration, dried in vacuo, and purifiedby flash chromatography (silica gel, 4×15 cm, eluted with 20% ether inCHCl₃) and recrystallized three times from methanol (MeOH) (0.49 g,15%).

[0303]¹H NMR(90 MHz, CDCl₃)δ8.01(d, J=9.9 Hz, 1H), 6.27-6.00(m, 3H),5.90 (d, J=2.6 Hz, 1H), 3.78(t, J=4.9 Hz, 4H), 2.67(t, J=4.9 Hz, 4H).IC₅₀(nM) DNA-PK Assay—100,000.

EXAMPLE 16 N¹-Morpholin-4-yl-4-nitrobenzene-1,3-diamine

[0304] A mixture of 5-chloro-2-nitrophenylamine (2.5 g, 14 mmol),4-aminomorpholine (4.4 g, 40 mmol), and K₂CO₃ (2.0 g, 14 mmol) in DMF(20 mL) was stirred at 120° C. for 18 h. The reaction mixture was cooledto room temperature and poured into water (150 mL). The crude solidproduct was collected by filtration, air dried, and purified by flashchromatography (silica gel, 4×15 cm, eluted with 20% ether in CHCl₃) toyield the product as a yellow solid (0.7 g, 20%).

[0305]¹H NMR (90 MHz, CDCl₃) δ8.06 (d, J=9.9 Hz, 1H), 6.28 (dd, J=9.9,2.6 Hz, 1H), 6.05 (br s, 2H), 5.97 (d, J=2.6 Hz, 1H), 3.85 (t, J=4.9 Hz,4H), 3.32 (t, J4.9 Hz, 4H).

[0306] IC₅₀(nM) DNA-PK Assay—1,000.

EXAMPLE 17 1-(3-Amino-4-nitrophenyl)-piperidin-4-ol

[0307] A mixture of 5-chloro-2-nitrophenylamine (1.3 g, 7.0 mmol),4-hydroxypiperidine (2.2 g, 20 mnmol), and K₂CO₃ (1.0 g, 7 mmnol) in DMF(20 mL) was stirred at 120° C. for 18 h. The reaction mixture was cooledto room temperature, poured into water (50 mL) and extracted with EtOAc(3×50 mL). The combined organic extracts were dried over sodium sulfate,concentrated in vacuo, and the oily residue was triturated with 70/30EtOAc/CHCl₃ (8 mL) to yield a yellow solid. The solid product wascollected by filtration, washed with 70:30 EtOAc/CHCl₃ (8 mL), and driedin vacuo (0.63 g, 38%).

[0308]¹H NMR (90 MHz, CDCl₃+D₂O) δ7.99 (d, 9.2 Hz, 1H), 6.26 (dd, J=9.9,2.6 Hz, 1H), 6.08 (d, J=2.6 Hz, 1H), 4.04-3.45 (m, 3H), 3.22-2.88 (m,2H), 2.02-1.25 (m, 4H).

EXAMPLE 18 2-Nitro-5-piperidin-1-yl-phenylamine

[0309] A mixture of 5-chloro-2-nitrophenylamine (5 g, 29 mmol),piperidine (2.9 mL, 29 mmol), and K₂CO₃ (4.0 g, 29 mmol) in DMF (20 mL)was stirred at 120° C. for 4 h. The reaction mixture was cooled to roomtemperature and poured into ice cold water (100 mL). The crude solidproduct was collected by filtration, air dried, and purified by flashchromatography (silica gel, 4×15 cm, eluted with CHCl₃) to yield theproduct as orange crystals (1.4 g, 22%). See, Aust. J. Chem., 47:247-262(1994).

[0310]¹H NMR (90 MHz, CDCl₃) δ7.90 (d, J=9.9 Hz, 1H, 6.25-6.00 (m, 3H),5.86 (d, J=2.6 Hz, 1H), 3.29 (br s, 4H), 1.58 (br s, 6H).

EXAMPLE 19 5-(4-Acetylpiperazin-1-yl)-2-nitrophenylamine

[0311] A mixture of 5-chloro-2-nitrophenylamine (5 g, 29 mmol),1-acetylpiperazine (29 mmol), and K₂CO₃ (4.0 g, 29 mmol) in DMF (20 mL)was stirred at 120° C. for 4 h. The reaction mixture was cooled to roomtemperature and poured into ice cold water (100 mL). The crude solidproduct was collected by filtration, air dried, and purified by flashchromatography (silica gel, 4×15 cm, eluted with CHCl₃) to yield theproduct as orange crystals.

[0312]¹H NMR (90 MHz, CDCl₃) δ11.46 (s, 1H), 9.59 (s, 1H), 7.35 (d,J=9.5 Hz, 1H), 6.46 (dd, J=9.5, 2.3 Hz, 1H), 6.28 (d, J=2.0 Hz, 1H),3.83 (t, J=4.6 Hz, 4H), 3.35 (t, J=4.9 Hz, 4H).

EXAMPLE 20 2-Nitro-5-piperazin-1-yl-phenylamine

[0313] A mixture of 5-chloro-2-nitrophenylamine (4.3 g, 25 mmol),piperazine (12.5 g, 145 mmol), and K₂CO₃ (4.0 g, 29 mmol) in DMF (50 mL)was stirred at 150° C. for 18 h. The reaction mixture was cooled to roomtemperature, solids were removed by filtration, and the filtrate wasconcentrated to a red residue. The residue was dissolved in EtOAc (100mL), washed with water (50 mL), dried with Na₂SO₄, and concentrated invacuo to afford the product as a yellow solid (3.9 g, 70%). See,Pharmazie, 39:747 (1984).

[0314]¹H NMR (90 MHz, d6-DMSO) δ7.89 (d, J=9.9 Hz, 1H), 7.09 (br s, 2H),6.35-6.20 (m, 2H), 3.33 ( t, J=4.6 Hz, 4H), 2.91 (t, J=4.8 Hz, 4H).

[0315] IC₅₀ (nM) DNA-PK Assay—10,000.

EXAMPLE 21 1-(3-Amino-4-nitrophenyl)-piperidin-3-ol

[0316] A mixture of 5-chloro-2-nitrophenylamine (1.3 g, 7.0 mmol),3-hydroxypiperidine hydrochloride (3.0 g, 20 mmol), and K₂CO₃ (3.0 g, 22mmol) in DMF (10 mL) was stirred at 120° C. for 18 h. The reactionmixture was cooled to room temperature, poured into water (100 mL) andextracted with EtOAc (3×100 mL). The combined organic extracts weredried over Na₂SO₄, concentrated in vacuo, and purified by flashchromatography (silica gel, 4×7.5 cm, eluted with 50:50 CHCl₃/EtOAc) toyield the product as an orange solid (0.89 g, 54%).

[0317]¹H NMR (90 MHz, d6-DMSO/CDCl₃) δ7.77 (d, J=9.0 Hz, 1H), 6.25-6.00(m, 2H), 3.82-3.25 (m, 3H), 3.02-2.62 (m, 2H), 1.90-1.12 (m, 4H).

[0318] IC₅₀ (nM) DNA-PK Assay—100,000.

EXAMPLE 21 N¹-(2-Morpholin-4-yl-ethyl)-4-nitrobenzene-1,3-diamine

[0319] A mixture of 5-chloro-2-nitrophenylamine (1.3 g, 7.0 mmol),2-(4-morpholinyl)-ethylamine (2.8 g, 20 mmol), and K₂CO₃ (3.0 g, 22mmol) in DMF (20 mL) was stirred at 110° C. for 48 h. The reactionmixture was cooled to room temperature, poured into water (200 mL) andextracted with dichloromethane (CH₂Cl₂) (250 mL). The combined organicextracts were washed with water (200 mL) and brine (200 mL), dried overNa₂SO₄, concentrated in vacuo, and purified by flash chromatography(silica gel, 4×15 cm, eluted with 90:10 EtOAc/MeOH) to yield the productas an orange solid (0.39 g, 20%).

[0320]¹H NMR (300 MHz, CDCl₃) δ7.96 (d, J=9.4 Hz, 1H), 6.23 (br s, 2H)6.00 (dd, J=9.5,2.1 Hz, 1H), 5.69 (d, J=1.9 Hz, 1H), 5.09 (s, 1H), 3.74(brt, J=4.3 Hz, 4H), 3.20 (br quartet, J=5.3 Hz, 2H), 2.65 (t, J=5.8 Hz,2H), 2.49 (br s, 4H).

[0321] IC₅₀ (nM) DNA-PK Assay—100,000.

EXAMPLE 23 5-(4-(2-Methoxyphenyl)-piperazin-1-yl]-2-nitrophenylamine

[0322] A mixture of 5-chloro-2-nitrophenylamine (1.3 g, 7.0 mmol),1-(2-methoxyphenyl)-piperazine hydrochloride (5.0 g, 20 mmol), and K₂CO₃(4.0 g, 29 mmol) in DMF (20 mL) was stirred at 110° C. for 18 h. Thereaction mixture was cooled to room temperature and poured into water(200 mL). The crude solid product was collected by filtration, airdried, and purified by trituration with CHCl₃ (1.3 g, 57%).

[0323]¹H NMR (90 MHZ, d6-DMSO/CDCl₃) δ8.23-7.83 (m, 2H), 7.17 (br s,2H), 6.93 (br s, 3H), 6.42-6.28 (m, 2H), 3.87 (s, 3H), 3.52-3.41, (m,4H), 3.20-3.12 (m, 4H).

[0324] IC₅₀ (nM) DNA-PK Assay—100,000.

EXAMPLE 24 5-(cis-2,6-Dimethylmorpholin-4-yl)-2-nitrophenylamine

[0325] A mixture of 5-chloro-2-nitrophenylamine (1.3 g, 7.0 mmol),cis-2,6-dimethylmorpholine (2.5 g, 20 mmol), and K₂CO₃ (3.0 g, 22 mmol)in DMF (20 mL) was stirred at 110° C. for 48 h. The reaction mixture wascooled to room temperature, poured into water (300 mL). The resultingcrude yellow solid was collected by vacuum filtration, air dried, andpurified by flash chromatography (silica gel, 4×7.5 cm, eluted with 95/5CHCl₃/ether) to yield the product as a yellow solid (1.2 g, 68%).

[0326]¹H NMR (90 MHz, CDCl₃) δ8.03 (d, J=9.9 Hz, 1H), 6.33-5.96 (m, 4H),3.82-3.55 (m, 4H), 2.58 (t, J=11.9 Hz, 2H), 1.28 (d, J=6.5 Hz, 6H). ¹³CNMR (75 MHz, CDCl₃) δ155.4, 147.0, 128.3, 125.7, 105.4, 98.6, 71.3,52.6, 18.9.

[0327] IC₅₀ (nM) DNA-PK Assay—100,000.

EXAMPLE 25 2-Nitro-5-(4-pyridin-2-yl-piperazin-1-yl)-phenylamine

[0328] A mixture of 5-chloro-2-nitrophenylamine (1.3 g, 7.0 mmol),1-(2-pyridyl)-piperazine (3.5 g, 20 mmol), and K₂CO₃ (3.0 g, 22 mmol) inDMF (20 mL) was stirred at 110° C. for 48 h. The reaction mixture wascooled to room temperature and poured into water. The crude yellow solidwas collected by vacuum filtration, air dried, and recrystallized fromCHCl₃ (0.36 g, 17%).

[0329]¹H NMR (90 MHz, CDCl₃) δ8.25-7.99 (m, 2H), 7.53-7.42 (m, 1H),6.74-6.61 (m, 2H), 6.36-5.95 (m, 4H), 3.73-3.46 (m, 8H). IC₅₀ (nM)DNA-PK Assay—100,000.

EXAMPLE 26 N¹-(3-Morpholin-4-yl-propyl)-4-nitrobenzene-1,3-diamine

[0330] A mixture of 5-chloro-2-nitrophenylamine (1.3 g, 7.0 mmol),3-(4-morpholinyl)-1-aminopropane (3.1 g, 20 nimol), and K₂CO₃ (3.0 g, 22mmol) in DMF (20 mL) was stirred at 110° C. for 18 h. The reactionmixture was cooled to room temperature, poured into water (200 mL) andextracted with CH₂Cl₂ (100 mL). The combined organic extracts werewashed with brine (50 mL), dried over Na₂SO₄, concentrated in vacuo, andpurified by flash chromatography (silica gel, 4×7.5 cm, eluted with90/10 EtOAc/MeOH) to yield the product as an orange solid (0.45 g, 23%).

[0331]¹H NMR (90 MHz, CDCl₃) δ7.93 (d, J=9.9 Hz, 1H), 6.23 (br s, 3H),5.92 (dd, J=9.2, 2.0 Hz, 1H), 5.65 (d, J=2.0 Hz, 1H), 3.74 (t, J=4.6 Hz,4H), 3.13 (br s, 2H), 2.58-2.44 (m, 6H), 1.80 (pentet, J=5.9 Hz, 2H).¹³C NMR (75 MHz, CDCl₃) δ154.2, 147.8, 128.5, 124.5, 105.9, 94.6, 67.1,57.5, 53.8, 42.8, 24.8.

[0332] IC₅₀ (nM) DNA-PK Assay—100,000.

EXAMPLE 27 2-Hydroxy-4-morpholin-4-yl-benzonitrile

[0333] A solution of 4-(4-morpholinyl)-salicylaldehyde oxime (0.9 g, 4mmol) pyridine (10 mL) was treated with trifluoroacetic anhydride (2.3mL, 16 mmol), stirred 18 h at room temperature, and concentrated invacuo at 80° C. The residue was dissolved in a mixture of EtOAc (15 mL)and CHCl₃ (10 mL), washed with water (2×20 mL), dried over Na₂SO₄, andconcentrated in vacuo. The product was purified by flash chromatography(silica gel, 4×7.5 cm, eluted with 80/20 CHCl₃/ether) to yield theproduct as a white solid (0.38 g, 42%).

[0334]¹H NMR (90 MHz, CDCl₃) δ7.30 (d, J=10.8 Hz, 1H), 6.50-6.40 (m,2H), 3.62, (t, J=5.4 Hz, 4H), 3.10 (t, J=5.4 Hz, 4H). ¹³C NMR (75 MHz,CDCl₃) δ161.7, 156.5, 134.7, 118.0, 107.8, 101.5, 90.2, 67.0, 48.3. LRMS(EI): m/e 204 (M⁺). IR (KBr): 3222, 2240 (CN), 1620 cm⁻¹.

[0335] IC₅₀ (nM) DNA-PK Assay—100,000.

EXAMPLE 28 (5-Morpholin-4-yl-2-nitrophenyl)-methanol

[0336] A mixture of 5-chloro-2-nitrobenzyl alcohol (1.9 g, 10.0 mmol),morpholine (0.9 mL, 10 mmol), and K₂CO₃ (1.4 g, 10 mmol) in DMF (20 mL)was stirred at 110° C. for 18 h. The reaction mixture was concentratedin vacuo, poured into aqueous sodium bicarbonate (NaHCO₃) (5% w/v, 25mL), and extracted with dichloromethane (25 mL). The organic extract waswashed with brine (25 mL), dried over Na₂SO₄, concentrated in vacuo, andpurified by flash chromatography (silica gel, 4×7.5 cm, eluted with90/10 EtOAc/MeOH) to yield a solid impure product. The residue wasrepurified in the same manner and the resulting solid was trituratedwith hexanes (10 mL) to afford the product as a yellow solid (5 mg, 2%).

[0337]¹H NMR (90 MHz, CDCl₃) δ8.14 (d, J=9.2 Hz, 1H), 7.03 (d, J=3.3 Hz,1H), 6.75 (dd, J=9.2, 3.3 Hz, 1H), 4.95 (d, J=4.0 Hz, 2H), 3.85 (t,J=4.9 Hz, 4H), 3.39 (t, J=4.9 Hz, 4H). ¹³C NMR (75 MHz, CDCl₃) δ154.7,139.9,138.1, 128.2, 113.3, 111.8, 66.4,63.7,47.3.

EXAMPLE 29 2-Hydroxy-4-morpholin-4-yl-benzoic acid

[0338] A solution of 2-hydroxy-4-morpholin-4-yl-benzonitrile (0.3 g, 1.5mmol) in concentrated HCl (10 mL) was stirred at reflux for 6 h, andconcentrated in vacuo. The residue was dissolved in an additionalportion of HCl (10 mL) and stirred under reflux for 6 h and concentratedto a dark solid. The solid was dissolved in aqueous sodium hydroxide(10% w/v, 2.5 mL) and treated with 6 N HCl (1 mL) to precipitate theproduct which was collected by filtration, washed with water (5 mL), anddried in vacuo at 40° C. (95 mg, 28%).

[0339]¹H NMR (90 MHz, d6-DMSO/D₂O): δ7.58 (d, J=9.2 Hz, 1H), 6.48 (d,J=9.2 Hz, 1H), 6.33 (br s, 1H), 3.70 (t, J=4.3 Hz, 4H), 3.24 (t, J=4.3Hz, 1H). ¹³C NMR (75 MHz, d6-DMSO/D₂O): δ171.5, 162.8, 156.2, 131.3,105.9, 102.6, 99.8, 65.9, 46.8. IR (KBr): 2975, 1626, 1570, 1515, 1240cm⁻¹.

[0340] IC₅₀ (nM) DNA-PK Assay—500.

EXAMPLE 30 2-Hydroxy-4-morpholin-4-yl-benzoic acid methyl ester

[0341] A solution of 2-hydroxy-4-morpholin-4-yl-benzonitrile (0.4 g, 2.0mmol) in concentrated HCl (20 mL) was stirred at reflux for 16 h, andconcentrated in vacuo to yield the corresponding carboxylic acid as acrude solid. The solid was suspended in MeOH (20 mL), treated with asolution of hydrogen chloride in dioxane (4 M, 10 mL), stirred at refluxfor 20 h, concentrated, treated again in the same manner and stirred 24h at reflux. The reaction mixture was concentrated, and the residue wasdissolved in EtOAc (20 mL), washed with aqueous sodium bicarbonate (5%,10 mL), dried over Na₂SO₄, and concentrated. The residue was purified byflash chromatography (silica gel, 4×7.5 cm, eluted with CHCl₃) to yieldthe product as a white solid (137 mg, 29%).

[0342]¹H NMR (90 MHz, CDCl₃): δ7.65 (d, J=10.7 Hz, 1H), 6.49-6.31 (m,2H), 3.87 (s, 3H), 3.80 (t, J=4.9 Hz, 4H), 3.24 (t, J=4.9 Hz, 4H). ¹³CNMR (75 MHz, CDCl₃): δ170.2, 163.3, 156.3. 130.9, 105.9, 103.3, 100.6,66.4, 51.5, 47.4.

[0343] IC₅₀ (nM) DNAPK Assay—500.

EXAMPLE 31 5-Morpholin-4-yl-2-nitro-benzamide

[0344] A mixture of 5-chloro-2-nitrobenzamide (2.0 g, 10.0 mmol),morpholine (2.6 mL, 30 mmol), and K₂CO₃ (4.1 g, 30 mmol) in DMF (20 ml)was stirred at 110° C. for 24 h. The reaction mixture was poured intowater (100 mL) and the resulting precipitate was collected by filtrationand recrystallized from ethanol to yield the product as a yellow solid(0.5 g, 20%).

[0345]¹H NMR (90 MHz, d6-DMSO): δ7.95 (d, J=9.2 Hz, 1H), 7.85 (br s,1H), 7.51 (br s, 1H), 7.06-6.86 (m, 2H), 3.72 (t, J=4.3 Hz, 4H), 3.38(t, J=4.3 Hz, 4H). ¹³C NMR (75 MHz, d6-DMSO): δ169.2, 154.8, 137.3,136.4, 127.4, 113.6, 113.0, 66.7, 47.7.

[0346] IC₅₀ (nM) DNA-PK Assay—100,000.

EXAMPLE 32 2-Hydroxy-4-morpholin-4-yl-benzaldehyde

[0347] DMF (10 mL) was treated dropwise with phosphorus oxychloride (2.3g, 15 mmol). The reaction was kept at 25° C. by cooling on ice. Thereaction mixture was treated with 3-(4-morpholinyl)phenol (2.5 g, 14mmol) in small portions, stirred for 30 min at room temperature, thenstirred at 100° C. for 8 h. After cooling, the mixture was poured intoaqueous sodium acetate (1 M, 40 mL) and 10 mL of water was added. Theresulting precipitate was collected by filtration, washed with water (10mL), air dried, and purified by flash chromatography (silica gel, 4×7.5cm, eluted with 90/10 CHCl₃/ether) to yield the product as a light graysolid (0.66 g, 23%). See U.S. Pat. No. 4,147,552.

[0348]¹H NMR (90 MHz, CDCl₃) δ11.45 (s, 1H), 9.59 (s, 1H), 7.36 (d,J=8.7 Hz, 1H), 6.45 (dd, J=8.7, 2.6 Hz, 1H), 6.27 (d, J=2.6 Hz, 1H),3.83 (t, J=5.4 Hz, 4H), 3.35 (t, J=4.9 Hz, 4H).

[0349]¹³C NMR (22.5 MHz, CDCl₃) δ193.0, 163.9, 156.9, 135.1, 113.3,124.0, 99.6, 66;4, 47.0. LRMS (EI): m/e 207 (M⁺), 149.

[0350] IC₅₀ (nM) DNA-PK Assay—400.

EXAMPLE 33 5-Morpholin-4-yl-2-nitro-phenol

[0351] A mixture of 2,4-dichloronitrobenzene (1.9 g, 10.0 mmol),morpholine (0.9 mL, 10 mmol), and K₂CO₃ (1.4 g, 10 mmol) in DMF (20 mL)was stirred at 110° C. for 18 h. The reaction mixture was poured intowater (100 mL) and extracted with CH₂Cl₂ (2×50 mL). The combined organicextracts were washed with brine (25 mL), dried over Na₂SO₄, concentratedin vacuo to yield 1.6 g of the crude intermediate, a mixture ofregioisomers: 4-(5-chloro-2-nitrophenyl)-morpholine:4-(3-chloro-4-nitrophenyl)-morpholine, 80:20.

[0352] The crude mixture (7 mmol) was suspended in water (85 mL) andtreated with sodium hydroxide (2.8 g, 70 mmol) and stirred at 150° C.for 16 h in a sealed container. After cooling to room temp, the mixturewas extracted with ether (2×50 mL) to remove unreacted startingmaterial. The aqueous layer was acidified with acetic acid (4.2 mL, 70mmol) to pH4 6 and then extracted with ether (3×100 mL). The etherextracts were dried over Na₂SO₄, concentrated in vacuo, and purified byflash chromatography (silica gel, 4×7.5 cm, eluted with 90/10CHCl₃/ether) to yield the faster-moving product as a yellow solid (45mg, 2%). The position of the morpholine was confirmed by 1D-Noe studies,irradiating both aromatic and morpholinyl protons. See, OrganicSyntheses Coll., Vol. II, 451 (1943).

[0353]¹H NMR (90 MHz, CDC13) δ11.1 (s, 1H), 7.87 (d, J=9.2 Hz, 1H),6,39-6.21 (m, 2H), 3.77 (t, J=4.9 Hz, 4H), 3.32 (t, J=4.9 Hz, 4H). ¹³CNMR (75 MHz, CDCl₃) δ157.9, 157.0, 127.2, 125.5, 106.6, 100.0, 66.3,47.0. LRMS (EI): 222 (M⁺), 94.

[0354] IC₅₀ (nM) DNA-PK Assay—2000.00

EXAMPLE 34 1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-ethanone

[0355] A solution of 2-hydroxy-4-morpholin-4-yl-benzaldehyde (1.0 g, 5mmol) in anhydrous tetrahydrofuran (20 mL) at −78° C. was treated withmethyilithium (1.5 M solution in peiitane, 7.1 mL, 11 mmol) at the rateof 0.5 mL/min. The reaction mixture was stirred at the same temperaturefor 1.5 h, allowed to warm to room temperature for 1 h, cooled to 0° C.,then quenched with sat. ammonium chloride (4 mL). The mixture wasextracted with ether (20 mL), and the ether layer was washed with 5%sodium bicarbonate (15 mL), dried over Na₂SO₄, and concentrated in vacuoto yield the desired intemniediate carbinol (1.1 g).

[0356] The crude carbinol intermediate (0.6 g, 2.7 mmol) was dissolvedin acetonitmLe (25 mL) and treated with manganese dioxide (2.34 g, 27mmol) and stirred for 48 h at room temperature. The solids were filteredoff through a pad of CELITE®, washed with acetonitmLe (20 mL), anddiscarded. The filtrate was concentrated in vacuo and purified by flashchromatography (silica gel, 4×7.5 cm, eluted with 90/10 CH₂Cl₂/ether) toyield the product as a white solid (208 mg, 25%).

[0357]¹H NMR (90 MHz, CDCl₃) δ7.55 (d, J=9.2 Hz, 1H), 6.43-6.28 (m, 2H),3.83 (t, J=4.6 Hz, 4H), 3.31 (t, J=4.9 Hz, 4H), 2.51 (s, 3H). ¹³CNMR (75MHz, CDCl₃) δ201.3, 165.0,156.6, 132.2,112.3,105.4, 100.6, 66.5, 47.2,25.6. LRMS (EI): m/e 221 (M⁺), 206, 163, 148.

[0358] IC₅₀ (riM) DNA-PK Assay—120.

EXAMPLE 35 1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-propan-1-one

[0359] Following the procedure for preparing Example 34,4-(4-morpholinyl) salicylaldehyde (1.0 g, 5 mmol) and ethylmagnesiumbromide (1.0 M solution in tetrahydrofuran, 11 mL, 11 mmol) wereconverted to the product (208 mg, 18%).

[0360]¹H NMR (300 MHz, CDCl₃): δ12.8 (s, 1H), 7.60 (d, J=9.1 Hz, 1H),6.37 (dd, J=9.1, 2.5 Hz, 1H), 6.28 (d, J=2.5 Hz, 1H), 3.82 (t, J=5.0 Hz,4H), 3.31 (t, J=5.0 Hz, 4H), 2.90 (q, J=7.3 Hz, 2H), 1.21 (t, J=7.3 Hz,3H). ¹³C NMR (75 MHz, CDCl₃): δ204.3, 165.0, 156.4, 131.4, 111.5, 105.4,100.7, 66.4, 47.2, 30.7, 8.8.

[0361] IC₅₀ (nM) DNA-PK Assay—120.

EXAMPLE 36 1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-3-methyl-butan-1one

[0362] Following the procedure for preparing Example 34,4-(4-morpholinyl)-salicylaldehyde (1.0 g, 5 mmol) and propylmagnesiumbromide (2.0 M solution in tetrahydrofuran, 5.5 mL, 11 mmol) wereconverted to the product (230 mg, 19%).

[0363]¹H NMR (300 MHz, CDCl₃): δ13.0 (s, 1H), 7.60 (d, J=9.2 Hz, 1H),6.37 (dd, J=9.1, 2.6 Hz, 1H), 6.28 (d, J=2.5 Hz, 1H), 3.82 (t, J=4.9 Hz,4H), 3.31 (t, J=4.9 Hz, 4H), 2.71 (d, J=7.0 Hz, 2H), 2.32-2.18 (m, 1H),0.99 (d, J=6.7 Hz, 6H). ¹³C NMR (75 MHz, CDCl₃): δ203.7, 165.2, 156.4,131.8, 112.2, 105.3, 100.7, 66.5, 47.2, 46.5, 26.1, 22.7.

[0364] IC₅₀ (nM) DNA-PK Assay—150.

EXAMPLE 37 1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-phenyl-methanone

[0365] Following the procedure for preparing Example 34,4-(4-morpholinyl)-salicylaldehyde (1.0 g, 5 mmol) and phenyllithium (1.8M solution in tetrahydrofuran, 6.0 mL, 11 mmol) were converted to theproduct (387 mg, 28%).

[0366]¹H NM (300 MHz, CDCl₃) δ12.74 (s, 1H), 7.64-7.43 (mn, 6H), 6.38(d, J=2.3 Hz, 1H), 6.33 (dd, J=9.1, 2.3 Hz, 1H), 3.84 (t, J=4.9 Hz, 4H),3.36 (t, J=4.9 Hz, 4H). ¹³C NMR (75 MHz, CDCl₃) δ198.9, 166.0,156.5,138,8, 135.2, 131.0, 128.7, 128.2, 111.3, 105.1, 100.5, 66.4, 47.1.

[0367] IC₅₀ (nM) DNA-PK Assay.--120.

EXAMPLE 382,2,2-Trifluoro-1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone

[0368] A solution of 4-(morpholin-4-yl)-salicylaldehyde (1 g, 4.8 mmol),in tetrahydrofuran (10 mL) was treated with a solution oftrimethyl(trifluoro-methyl) silane (0.5 M in tetrahydrofuran, 21 mL,10.5 mmol) and tetra-n-butylammonium fluoride (20 mg, catalytic). Afterstirring for 3 h at room temperature, the reaction mixture was pouredinto ether (100 mL), washed with brine (25 mL), saturated aqueous NaHCO₃(25 mL), brine (25 mL), dried over magnesium sulfate, and concentratedto a syrup that solidified upon standing (1.35 g crude carbinol).

[0369] The crude carbinol (1.16 g, 4.18 mmol) was dissolved in CH₂Cl₂and the solution was treated withoxo-bis-trifluoromethyl-1,3-dihydro-1(5-1,2-benziodoxol-1-ol (3.36 g,8.36 mmol) and stirred for 1 h at room temperature. The reaction mixturewas filtered through a plug of silica gel (150 g in a 250 mL frittedfunnel) and the silica gel was washed with CH₂Cl₂. The combinedfiltrates were concentrated to a syrup and treated with hexanes (50 mL).The resulting precipitate was removed by filtration and discarded. Thefiltrate was concentrated and purified by flash chromatography (silicagel, 4×15 cm, eluted with 5-15% ether in hexanes) to yield the productas a yellow solid (220 mg, 19%). M.P.: 81-82° C. See, J. Org Chem.56:984 (1991); J. Am. Chem. Soc. 115:6078 (1993).

[0370]¹H NMR (300 MHz, CDCl₃) δ11.62 (s, 1H), 7.76 (d, J=9.2 Hz, 1H),6.42 (d, J=9.2 Hz, 1H), 6.26 (s, 1H), 3.81 (t, J=4.9 Hz, 4H), 3.41 (t,J=4.9 Hz, 4H). ¹³C NMR (75 MHz, CDCl₃) δ180.6 (q, JC-F=34 Hz), 167.4,157.5, 132.3, 117.2 (q, JC-F=288 Hz), 106.3, 106.0, 99.6, 66.3, 46.7.LRMS (EI): 275 (M+), 206, 148.

[0371] IC₅₀ (nM) DNA-PK Assay—250.

EXAMPLE 39 4-Amino-2-morpholin-4-yl-pyrimidine-5-carboxylic acid

[0372] A mixture of 4-amino-5-carboxy-2-ethylmercaptopyrimidine (0.35 g,1.8 mmol) and morpholine (0.5 mL, 10 mmol) was warmed at 80° C. for 4 dand concentrated in vacuo. The residue was purified by flashchromatography (silica gel, 4×7.5 cm, eluted with 97.25/2.5/0.25acetonitrile/water/acetic acid) to yield the product as a white solid(34 mg, 30%).

[0373]¹H NMR (300 MHz, d6-DMSO) δ8.49 (s, 1H), 7.65 (br s, 11), 7.35,(br s, 1H), 3.75 (t, J=4.4 Hz, 4H), 3.63 (t, J=4.4 Hz, 4H). ¹³C NMR (75MHz, d6-DMSO) δ167.5, 162.9, 161.5, 160.5, 95.5, 65.8, 43.7.

[0374] IC₅₀ (nM) DNAPK Assay—100,000.

EXAMPLE 40 1-(5-Bromo-2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone

[0375] A solution of 4-(4-morpholinyl)-2-hydroxyacetophenone (0.38 g,1.3 numol) in CHCl₃ was added to a suspension of cupric bromide (0.46 g,2.0 mmol) in EtOAc (10 mL) and stirred for 16 h at reflux. The reactionmixture was treated with more cupric bromide (0.39 g, 1.7 mmol) andstirred 20 h at reflux. Solids were filtered off, and the filtrate wasconcentrated and purified by flash chromatography (silica gel, 4×7.5 cm,eluted with 15-20% ether in hexanes) to yield the product as a yellowsolid (111 mg, 28%).

[0376]¹H NMR (300 MHz, CDCl₃) δ12.39 (s, 1H), 7.87 (s, 1H), 6.53 (s,1H), 3.87 (t, J=4.5 Hz, 4H), 3.15(, J=4.5 Hz, 4H), 2.56 (s, 3H).

[0377] IC₅₀ (nM) DNA-PK Assay—100,000.

EXAMPLE 41 1-(3-Bromo-2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone

[0378] A solution of 4-(4-morpholinyl)-2-hydroxyacetophenone (0.17 g,0.6 mmol) in MeOH was treated with cupric bromide (0.20 g, 0.9 mmol) andstirred for 16 h at reflux. The reaction mixture was treated with morecupric bromide (0.39 g, 1.7 mmol) and stirred 20 h at reflux. The solidswere filtered off, and the filtrate was concentrated and purified byflash chromatography (silica gel, 4×7.5 cm, eluted with 10% ether inhexanes) to yield the product as a yellow syrup (62 mg, 34%).

[0379]¹H NMR (300 MHz, CDCl₃) δ7.66 (d, J=8.5 Hz, 1H), 6.56 (d, J=8.5Hz, 1 H), 3.88 (t, J=4.5 Hz, 4H), 3.20 (t, J=4.5 Hz, 4H). ¹³C NMR (75MHz, CDCl₃) δ202.5, 161.0, 157.5, 130.5, 115.9, 110.6, 106.5, 66.9,51.5, 26.0.

[0380] IC₅₀ (nM) DNA-PK Assay—100,000.

EXAMPLE 42 A:1-(3,5-Dichloro-2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone B:1-(3-Chloro-2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone

[0381] A solution of 4-(4-morpholinyl)-2-hydroxyacetophenone (0.21 g,0.7 mmol) in glacial acetic acid (2 mL) was treated with chlorine gasand stirred 18 h at room temperature. The entire mixture was poured intowater (30 mL) and extracted with ether (2×25 mL). The combined organicextracts were washed with 5% NaHCO₃ (2×30 mL) and brine (30 mL), driedwith Na₂SO₄, concentrated in vacuo. The residue was purified by flashchromatography (silica gel, 4×7.5 cm, eluted with 10-20% EtOAc inhexanes) to yield A (faster-eluting band, 40 mg, 18%) and B(slower-eluting band, 60 mg, 27%). See, ACCUFLUOR® brochure fromAllied-Signal Corp.

[0382]¹H NMR (300 MHz, CDCl₃) δ12.97 (s, 1H), 7.66 (s, 1H), 3.85 (t,J=4.5 Hz, 4H), 3.36 (t, J=4.5 Hz, 4H), 2.60 (s, 3H). LRMS (EI): 291/289(M⁺), 254, 231, 216.

[0383] IC₅₀ (nM) DNA-PK Assay—100,000.

[0384]¹H NMR (300 MHz, CDCl₃) δ13.19 (s, 1H), 7.62 (d, J=8.9 Hz, 1H),6.56 (d, J=8.9 Hz, 1H), 3.39 (t, J=4.6 Hz, 4H), 3.22 (t, J=4.5 Hz, 4H),2.60 (s, 3H). LRMS (EI): 257/255 (M⁺), 199, 182.

[0385] IC₅₀ (p,1) DNA-PK Assay—14,000.

EXAMPLE 43 C: 1-(5-Fluoro-2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone D:1-(3-Fluoro-2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone

[0386] A solution of 4-(4-morpholinyl)-2-hydroxyacetophenone (1.0 g, 4.5mmol) in acetonitmLe (20 mL) was treated with ACCUFLUOR® NF-Th (50% w/won alumina, 5.8 g, 9 mmol) and warmed at 40° C. for 48 h. The solidswere removed by filtration, and the filtrate was concentrated, dissolvedin CH₂Cl₂ (50 mL), and washed with 5% NaHCO₃ (50 mL). The CH₂Cl₂ layerwas dried over Na₂SO₄, concentrated, and the residue was purified byflash chromatography (silica gel, 4×7.5 cm, eluted with 15% EtOAc inhexanes) to yield C (faster-eluting band, 83 mg, 8%) and D(slower-eluting band, 95 mg, 9%).

[0387]¹H NMR (300 MHz, CDCl₃): δ12.41 (s, 1H), 7.29 (d, JH-F 13.5 Hz,1H), 6.34 (d, JH-F=7.4 Hz, 1H), 3.85 (t, J=4.5 Hz, 4H), 3.26 (t, J=4.5Hz, 4H), 2.50 (s, 3H). ¹³C NMR (75 MHz, CDCl₃): δ202.4, 161.6, 148.2 (d,JC-F=9 Hz), 148.1 (d, JC-F=240 Hz), 117.5 (d, JC-F=23 Hz), 112.9, 106.5,67.6, 50.8, 27.0. LRMS (EI): m/e 239 (M⁺), 224, 167, 155.

[0388] IC⁵⁰ (nM) DNA-PK Assay—1,500.

[0389]¹H NMR (300 MHz, CDCl₃): δ12.51 (s, 3H), 7.44 (dd, JH-H=8.8 Hz,JH-F=1.6 Hz, 1H), 6.41 (JH-H=8.8 Hz, JH-F=7.7 Hz, 1H), 3.88 (t, J=4.5Hz, 4H), 3.30 (t, J=4.5 Hz, 4H). ¹³C NMR (75 MHz, CDCl₃): δ202.6, 152.4(d, JC-F=13 Hz), 145.4 (d, JC-F=4 Hz), 142.2 (d, JC-F=244 Hz), 126.3,115.1, 107.4, 67.0, 49.9, 26.0. LRMS (EI): m/e 239 (M⁺), 224, 196, 167,155, 127.

[0390] IC₅₀ (nM) DNA-PK Assay—240.00

EXAMPLE 44 1-(2-Hydroxy-4-(tetrahydropyran-4-yloxy)-phenyl]-ethanone

[0391] A mixture of tetrahydro-4H-pyran-4-ol (1.53 g, 15 mmol),2,4-dihydroxyacetophenone (1.52 g, 10 mmol), and triphenyiphosphine,polymer-bound (5 g, 3 mmol P/g resin, 15 mmol) in CH₂Cl₂ (150 mL) wastreated with diethyilazodicarboxylate (2.61 g, 15 mmol) and stirred at20° C. for 20 h. The reaction mixture was treated with ether (200 mL)and the resin was filtered off and discarded. The filtrate wasconcentrated irn vacuo and purified by flash chromatography (silica gel,4×7.5 cm, eluted with 25% ether in hexanes) to yield the white solidproduct (0.74 g, 31%).

[0392] M.P.: 99-104° C. ¹H NMR (90 MHz, CDCl₃) δ12.71 (s, 1H), 7.63 (d,J=9.9 Hz, 1H), 6.50-6.40 (m, 2H), 4.60-4.50 (m, 1H), 4.11-3.85 (m, 2H),3.67-3.45 (m, 2H), 2.55 (s, 3H), 2.00-1.74 (m, 4H). ¹³C NMR (75 MHz,CDCl₃) δ200.2, 165.1, 163.9, 132.3, 114.0, 108.5, 102.4, 71.9, 64.8,31.5, 25.8.

[0393] IC₅₀ (nM) DNA-PK Assay—100,000.

EXAMPLE 45 5-(Morpholin-4-yl)-1,3-dihydro-benzimidazol-2-one

[0394] A solution of 5-morpholin-4-yl-2-nitro-phenylamine (4.0 g, 0.02mol) in acetic acid (100 mL) and ethanol (100 mL) was treated withhydrogen gas (60 psi) for 30 min in the presence of palladium on carboncatalyst (0.5 g, 10% Pd). The catalyst was removed by filtration throughCELITE®, and the filtrate was concentrated to a syrup and dried in vacuoto afford the intermediate 4-morpholin-4-yl-benzene-1,2-diamine as acrude green solid (3.2 g).

[0395] A solution of crude 4-morpholin-4-yl-benzene-1,2-diamine (0.75 g,3.9 mmol) in tetrahydroflran (4 mL) was treated dropwise with a solutionof carbonyldiimidazole (0.76 g, 4.7 mmol) in tetrahydrofuran (12 mL).The reaction mixture was stirred for 16 h at room temperature, and thesolid product was collected by filtration and recrystallized fromethanol to afford the white solid product (0.47 g, 55%). See, J. Am.Chem., Soc., 118:4018 (1996).

[0396]¹H NMR (90 MHz, d6-DMSO): δ11.17 (s, 1H), 11.05 (s, 1H), 7.55 (d,J=9.2 Hz, 1H), 7.32-7.24 (m, 2H), 4.46 (t, J=4.3 Hz, 4H), 3.70 (t, J=4.6Hz, 4H). ¹³C NMR (75 MHz, d6-DMSO): δ155.4, 146.2, 130.3, 123.4, 108.9,108.4, 97.5, 66.1, 50.2.

[0397] IC₅₀ (nM) DNA-PK Assay—100,000.

EXAMPLE 46 A: 2-Methoxy-4-morpholin-4-yl-benzaldehyde B:4-Methoxy-6-morpholin-4-yl-benzene1,3-dicarbaldehyde

[0398] A solution of m-anisidine (11.2 mL, 0.1 mol), chloroethyl ether(11.7 mL, 0.1 mol), and diisopropylethylarine (35 mL, 0.2 mol) intoluene (100 mL) was stirred at reflux for 72 h. The precipitate wasremoved by filtration and washed with toluene (100 mL). The combinedtoluene solutions were concentrated in vacuo and purified bydistillation (120-130° C., 0.2 Torr) to afford the intermediate4-(3-methoxyphenyl)-morpholine (14.9 g, 77%).

[0399] DMF (10 mL) was treated dropwise with phosphorus oxychloride (1.3g, 14 mmol). The reaction was kept at 25° C. bycooling on ice. Thereaction mixture was treated with 4-(3-methoxyphenyl)-morpholine (2.5 g,13 mmol) in small portions, stirred for 30 min at room temperature, thenstirred at 100° C. for 4 h. After cooling, the mixture was poured intoaqueous sodium acetate (1 M, 40 mL) and 25 mL of water was added. Theresulting precipitate was collected by filtration, washed with water (10mL), air dried, and purified by flash chromatography (silica gel, 4×15cm, eluted with 80/20 CHCl₃/ether) to yield A and B as white solids: A:(1.0 g, 35%), B: (0.3 g, 9%).

[0400]¹H NMR (90 MHz, CDCl₃): δ10.21 (s, 1H), 7.75 (d, J=8.6 Hz, 1H),6.48 (dd, J=8.6, 1.7 Hz, 1H), 6.30 (s, 1H), 3.90-3.80 (m, 7H), 3.32 (t,J=4.9 Hz, 4H). ¹³C NMR (75 MHz, CDCl₃): δ187.5, 164.2, 156.1, 1t30.0,117.5, 106.4, 96.0, 65.0, 54.9, 46.3.

[0401] IC₅₀ (nM) DNA-PK Assay—10,000.

[0402]¹H NMR (90 MHz, CDCl₃): δ10.26 (s, 1H), 9.93 (s, 1H), 8.27 (s,1H), 6.40 (s, 1H), 3.99-3.88 (m, 7H), 3.24 (t, J=4.6 Hz, 4H). ¹³ C NMR(75 MHz, CDCl₃): δ188.2, 187.3, 166.1, 159.8, 137.7, 120.8, 119.3, 99.8,66.2, 57.7, 52.5.

EXAMPLE 47 2-Hydroxy-5-morpholin-4-yl-benzoic acid methyl ester

[0403] Step 1: A solution of methyl 5-nitrosalicylate (10.0 g, 50.7mmol) and imidazole (3.8 g, 55.8 mol) in DMF (50 mL) at 0° C. wastreated with t-butyl-chloro-dimethyl-.silane and stirred 16 h at roomtemperature. The reaction mixture was poured into water (100 mL) andextracted with ether (3×50 mL). The combined extracts were washed withwater (4×100 mL) and brine (50 mL), dried over magnesium sulfate, andconcentrated. The crude residue was purified by flash chromatography(silica gel, 4×15 cm, eluted with 95/5 hexanes/EtOAc) to yield2-(tert-butyl-dimethyl-silanyloxy)-5-nitro-benzoic acid methyl ester asa white solid (4.9 g, 31%).

[0404] Step 2: A solution of2-(tert-butyl-dimethyl-silanyloxy)-5-Nitro-benzoic acid methyl ester(4.9 g, 16 mmol) in ethanol (100 mL) was treated with hydrogen gas (60psi) for 48 h in the presence of palladium on carbon catalyst (0.31 g,10% Pd). The catalyst was removed by filtration through CELITE®, and thefiltrate was concentrated to a syrup and dried in vacuo to afford theintermediate 5-amino-2-(tert-butyl-dimethyl-silanyloxy)-benzoic acidmethyl ester as a dark brown solid (4.52 g, 99%).

[0405] Step 3: A solutionof5-amino-2-(tert-butyl-dimethyl-silanyloxy)-benzoic acid methyl ester(4.5 g, 16 mmol), 2-chloroethyl ether (1.9 mL, 16.2 mmol), anddiisopropylethylamine (5.6 mL, 32.1 mmol) in DMF (50 mL) was stirred at100° C. for 48 h. The reaction mixture was poured into 10% aqueousNaHCO₃ (100 mL) and extracted with ether (200 mL). The organic layer waswashed with water (5×100 mL) and brine (100 mL), dried over magnesiumsulfate (MgSO₄), and concentrated to a red syrup. The residue waspurified by flash chromatography (silica gel, 4×15 cm, eluted with 9/1hexanes/EtOAc) to yield2-(tert-butyl-dimethyl-silanyloxy)-5-morpholin-4-yl-benzoic acid methylester (1.0 g, 18%).

[0406] Step 4: A solution of2-(tert-butyl-dimethyl-silanyloxy)-5-morpholin-4-yl-benzoic acid methylester (1.0 g, 2.8 mmol) in tetrahydrofuran (35 mL) was treated withtetrabutylammonium fluoride (3.55 g, 11.25 mmol) and stirred for 2 h atroom temperature. The reaction mixture was concentrated and purified byflash chromatography (silica gel, 4×7.5 cm, eluted with 80/20hexanes/EtOAc) to yield the final product as a yellow solid (206 mg,31%).

[0407]¹H NMR (300 MHz, CDCl₃): δ10.40 (s, 1H), 7.34 (d, J=2.7 Hz, 1H),7.16 (dd, J=9.0, 2.7 Hz, 1H), 6,94 (d, J=9.0 Hz, 1H), 3.95 (s, 3H),3.89-3.86 (m, 4H), 3.08-3.05 (m, 4H). ¹³C NMR (75 MHz, CDCl₃): δ170.4,156.3, 144.3, 126.2, 118.3, 116.4, 112.2, 67.0, 52.1, 50.9. LRMS (EI):m/z 237 (M⁺), 205, 177, 147.

[0408] IC₅₀ (nM) DNA-PK Assay—40,000.

EXAMPLE 48 2-((Hydroxyimino)methyl)-5-morpholin-4-yl-phenol

[0409] A solution of 2-hydroxy-4-mo01pholin-4-yl-ben zaldehyde (0.135 g,0.65 mmol) in pyridine (5 mL) was treated with hydroxylaminehydrochloride (45 mg, 0.65 mmol) and stirred for 30 min at roomtemperature. The reaction mixture was concentrated in vacuo and theresidue was partitioned between EtOAc and 5% sodium bicarbonate. Theorganic layer was dried over Na₂SO₄ and concentrated in vacuo to affordthe product as a pale yellow solid (51 mg, 35%).

[0410]¹H NMR (90 MHz, CDCl₃) δ8.04 (s, 1H), 7.34 (s, 1H), 6.97 (d, J=9.2Hz, 1H), 6.44-6.32 (m, 3H), 3.76 (t, J=4.9 Hz, 4H), 3.13 (t, J==4.9 Hz,4H).

[0411] IC₅₀ (nM) DNA-PK Assay—1,000.

EXAMPLE 49 2-Hydrazonomethyl-5-morpholin-4-yl-phenol

[0412] A solution of 2-hydroxy-4-morpholin-4-yl-benzaldehyde (0.135 g,0.65 mmol) in pyridine (5 mL) was treated with hydrazine hydrate (33 mg,0.65 mmol) and stirred for 3 h at room temperature. The reaction mixturewas concentrated in vacuo and the residue was partitioned between EtOAc.and 5% sodium bicarbonate. The organic layer was dried over Na₂SO₄ andconcentrated in vacuo to afford the product as a pale yellow solid (96mg, 67%)

[0413]¹H NMR (90 MHz, CDCl₃) δ7.80 (s, 1H), 6.97 (d, J=9.3 Hz, 1H),6.44-6.32 (m, 3H), 5.24 (br s, 2H), 3.83 (t, J=4.9 Hz, 4H), 3.18 (t,J=4.9 Hz, 4H).

[0414] IC₅₀ (nM) DNA-PK Assay—1,000.

EXAMPLE 50 2-Hydroxy-4-((1-morpholin-4-yl-methanoyl)amino]-benzoic acid

[0415] A solution of 4-amino-2-hydroxy-benzoic acid methyl ester (1.0 g,6 mmol) and triethylamine (0.8 mL, 6 mmol) in CHCl₃ (25 mL) was treatedwith morpholine-4-carbonyl chloride (4.2 mL, 48 mmol) and stirred atreflux for 72 h. After cooling to room temperature, the solution waswashed with 5% aqueous hydrochloric acid (25 mL), saturated aqueoussodium bicarbonate (25 mL), and brine (25 mL), dried over Na₂SO₄, andconcentrated to an orange solid. The crude solid was triturated withCHCl₃ (10 mL) to afford the product as an off-white solid (0.25 g, 15%).

[0416]¹H NMR (300 MHz, d6-DMSO): δ10.47 (s, 1H), 8.17 (s, 1H), 7.42 (d,J=8,8 Hz, 1H), 6.90 (s, 1H), 6.81 (dd, J=8.8, 1.7 Hz, 1H), 3.62 (s, 3H),3.43 (t, J=4.5H, 4H), 3.27 (t, J=4.5 Hz, 4H).

[0417] IC₅₀ (mM) DNA-PK Assay—100,000.

EXAMPLE 51 2-Hydroxy-4-morpholin-4-ylmethyl-benzoic acid methyl esterhydrochloride

[0418] Step 1: A solution of 4-methylsalicylic acid (75 g, 0.49 mmol) inMeOH (500 mL) at 0° C. was treated with dicyclohexylcarbodiimide (101 g,0.49 mmol) and the mixture was stirred at room temperature for 2 h. Theprecipitated dicyclohexylurea was removed by filtration and the filtratewas concentrated in vacuo. The crude residue was purified bydistillation (110° C. bath temp, 0.5 mm Hg) to afford 4-methylsalicylicacid methyl ester

[0419] Step 2: A refluxing solution of 4-methylsalicylic acid methylester (5.1 g, 31.3 mmol) in carbon tetrachloride (50 mL) was treateddropwise with a solution of bromine (1.6 mL, 31.3 mmol) in carbontetrachloride (5 mL) while under illumination by a 200 W incandescentlight bulb. The reaction mixture was stirred for an additional 30 min atreflux and concentrated in vacuo. The residue was dissolved in EtOAc (50mL) and washed with aqueous sodium thiosulfate (5%, 50 mL), water (50mL), and brine (50 mL). The organic layer was dried over Na₂SO₄,concentrated, and the product 4-bromomethylsalicylic acid methyl esterwas isolated by recrystallization from heptane (1.6 g, 21%).

[0420] Step 3: A solution of 4-bromomethylsalicylic acid methyl ester(245 mg, 1 mmol) and triethylamine (0.139 mL, 1 mmol) in acetonitmLe (5mL) was treated with morpholine (0.087 mL, 1 mmol) and stirred at roomtemperature for 2 h. The reaction mixture was poured into saturatedaqueous sodium bicarbonate (50 mL) and extracted with EtOAc (3×25 mL).The combined organic extracts were washed with water (25 mL) and brine(25 mL), dried with Na₂SO₄, and concentrated in vacuo. The residue wasdissolved in ether (30 mL), cooled to 0° C., and treated with HCl gas.An equal volume of heptane was added and the crude solid product wascollected by filtration, washed with heptane, and dried in vacuo. Halfof the solid was purified by recrystallization from EtOAc/MeOH/heptaneto yield the product 2-hydroxy-4-morpholin-4-yl-methyl-benzoic acidmethyl ester hydrochloride (49 mg, 17%). M.P.: 213-214° C.

[0421]¹H NMR (300 MHz, d6-DMSO): δ11.65 (bs, 1H), 10.59 (s, 1H), 7.82(d, J=8.1 Hz, 1H), 7.32 (s, 1H), 7.22 (d, J=8.1 Hz, 1H), 4.32 (d, J=5.1Hz, 2H), 3.95-3.70 (m, 4H), 3.90 (s, 3H), 3.25-3.00 (m, 4H). ¹³C NMR (75MHz, d6-DMSO): δ168.6, 159.6, 136.8, 130.4, 122.2, 120.4, 114.2, 63.0,58.1, 52.6, 50.8. Elemental analysis: Calc'd: C, 54.26; H, 6.31%; N,4.87%, Found: C, 54.19%; H, 6.20%; N, 4.65%.

[0422] IC₅₀ (nM) DNA-PK Assay—100,000.

EXAMPLE 52 2-Hydroxy-4-morpholin-4-ylmethyl-benzoic acidtrifluoroacetate

[0423] A solution of 2-hydroxy-4-morpholin-4-ylmethyl-benzoic acidmethyl ester hydrochloride (100 mg, 0.35 mmol) in MeOH (2 mL) wastreated with 1 M sodium hydroxide (1.4 mL, 1.4 mmol) and stirred at 50°C. for 18 h. The reaction mixture was concentrated in vacuo to ¼ theoriginal volume, treated with 1 M hydrochloric acid (0.7 mL, 6.7 mmol).The solution was purified by HPLC on a 2.1×25 cm Vydac C18 Protein andPeptide column, 20 mL/min, with a gradient as follows (0-5 min: 100%water, 25 min: 30% acetonitrile/70% water. Both solvents contained 0.05%trifluoroacetic acid). The product fractions were combined andlyophilized to a white powder.

[0424] M.P.: 138-146° C. ¹H NMR (300 MHz, d6-DMSO) δ7.87 (d, J=8.0 Hz,1H), 7.11 (bs, 1H), 7.03 (dd, J=8.0Hz, 1H), 4.30 (s, 2H), 3.78 (bs, 4H),3.15 (bs, 4H). Elemental analysis: Calc'd for C₁₂H₁₅NO₄.1.1 C₂HF₃O₂: C,47.03; H, 4.47%; N, 3.86%. Found: C, 47.03%; H, 4.37%; N, 3.87%.

[0425] IC₅₀ (nM) DNA-PK Assay—100,000.

EXAMPLE 53 2-Hydroxy-4-morpholin-4-ylmethyl benzoic acid hydrochloride

[0426] A solution of 2-hydroxy-4-morpholin-4-ylmethyl-benzoic acidmethyl ester hydrochloride (75 mg, 0.26 mmol) in MeOH (0.5 mL) wastreated with ammonium hydroxide (1 mL) and stirred in a sealed vial for2 h at 60° C. After cooling, the reaction mixture was concentrated undera stream of nitrogen to approximately ¼ the original volume and wastreated with 1 M HCl (0.25 mL, 0.25 mmol). The solid that formed wascollected by filtration and recrystallized from water to yield the whitecrystalline product.

[0427]¹H NMR (300 MHz, d6-DMSO: δ13.21 (s, 1H), 10.95 (br s, 1H), 8.51(s, 1H), 8.05 (s, 1H), 7.92 (d, J=8.1 Hz, 1H), 7.18-7.01 (m, 3H), 4.28(s, 2H), 3.95-3.72 (m, 4H), 3.25-3.06 (m, 4H). Elemental analysis:Calc'd for C₁₂H₁₆N₂O₃HCl: C, 52.85; H, 6.28%; N, 10.27%. Found: C,52.47%; H, 6.16%; N, 10.10%.

[0428] IC₅₀ (nM) DNA-PK Assay—100,000.

EXAMPLE 54 4-Amino-2-hydroxy-benzoic acid methyl ester

[0429] Esterification Procedure

[0430] To a stirred solution of 4-amino salicylic acid (110.0 g; 718mmol) in dry MeOH (3025 mL) was slowly added concentrated sulfuric acid(187 mL; 3.5 mmol) via pipette at room temperature under nitrogenatmosphere. The resulting solution was heated to reflux for 20 hoursthen allowed to cool to room temperature. The reaction was concentratedat reduced pressure to about {fraction (1/4)} the original volume, thenneutralized by the careful addition of saturated aqueous sodiumbicarbonate to pH 7-8. The resulting precipitate was collected on aBuchner funnel with suction, washed with water, and allowed to air dry.The resulting gray solid was dissolved in EtOAc (1.5 L) and treated withdecolorizing charcoal. Filtration and recrystallization (EtOAc/hexanesor MeOH/water (1:3)) provided the ester as an off white crystal (94.5 g;79%).

[0431]¹H NMR (CDCl₃, 400 MHz): δ10.95 (s, 1H), 7.59 (dd, 1H), 6.14 (s,1H), 6.12 (d, 1H), 4.16 (br s, 2H), 3.85 (s, 3H).

EXAMPLE 55 2-Hydroxy-4-morpholin-4-yl-benzoic acid methyl ester

[0432] Morpholine Cyclization Procedure

[0433] A stirred suspension of free aniline (50.0 g; 299 mmol), Hunig'sbase (104.3 mL; 598 mmol), chloro-ethyl ether (35.1 mL; 299 mmol) andsodium iodide (89.75 g; 598 mmol) in dry toluene (450 mL) was heated toreflux under a nitrogen atmosphere for 5 days. After cooling to roomtemperature, the reaction was quenched with cold aqueous citric acid(10%) and extracted with EtOAc (3×500 mL). Combined organic extractswere washed with 5% aqueous sodium bicarbonate and brine then dried(Na₂SO₄), decolorized (activated charcoal), filtered and concentrated invacuo. The residue was purified via flash chromatography (5:1hexanes/EtOAc on silica gel) to provide the morpholine as a whitecrystalline product (26.0 g; 37%).

[0434]¹H NMR (CDCl₃, 400 MHz): δ10.86 (s, 1H), 7.65 (d, 1H), 6.36 (dd,1H), 6.31 (d, 1H), 3.87 (s, 3H), 3.79 (t, 4H), 3.25 (t, 4H).

EXAMPLE 56 2-Hydroxy-N-methyl-4-morpholin-4-yl-benzamide

[0435] Weinreb Amidation Procedure

[0436] To a cooled (0° C.), stirred solution of methylamine in drytetrahydrofuran (1.27 mL of 2.0 M solution; 2.52 mmol) was slowly addeda solution of trimethylaluminum in dry toluene (1.27 mL of 2.0 Msolution; 2.52 mmol) via syringe under a nitrogen atmosphere. Theresulting solution was allowed to warm to room temperature and stir for2 hours. To the resulting solution, a solution of the2-hydroxy-4-morpholin-4-yl-benzoic acid methyl ester (100 mg; 0.42 mmol)in dry toluene (4.0 mL) was added via cannula and the resulting solutionwas heated to reflux for 2 hours. After cooling to room temperature, thereaction was quenched with the careful addition of 1 N aqueoushydrochloric acid to pH 4. The mixture then was extracted with EtOAc(3×10 mL), and the combined organic layers washed with brine, dried(Na₂SO₄), filtered and concentrated in vacuo. The residue wasrecrystallized from CH₂Cl₂- hexanes to provide the amide (93 mg; 93%).

[0437]¹NMR (CDCl₃, 400 MHz, mixture of rotomers): δ12.60 (s, 111), 7.20(d, 1H), 6.38-6.33 (m, 2H), 6.14 (br s, 1H), 3.82 (t, 4H), 3.23 (t, 4H),3H). LRMS (Electrospray, negative): Da/e 235.2 (m−1).

EXAMPLE 571-(2.Hydroxy-4-morpholin-4-yl-phenyl)-1-morpholin-4-yl-methanone

[0438] Prepared via the Weinreb amnidation procedure of Example 56.

[0439]¹H NMR (CDCl3, 400 MHz): δ10.45 (s, 1H), 7.14 (d, 1H), 6.42 (d,1H), 6.35 (dd, 1H), 3.83 (t, 4H), 3.73 (s, 8H), 3.23 (t, 4H). LRMS(Electrospray, negative): Da/e 291.3 (m−1).

EXAMPLE 58 2-Hydroxy-4-morpholin-4-yl-benzamide

[0440] Prepared via the Weinreb amidation procedure of Example 56.

[0441]¹H NMR (CDCl₃, 400 MHz): δ7.33 (d, 1H), 6.45 (dd, 1H), 6.37 (d,1H), 6.15 (br s, 2H), 3.83 (t, 4H), 3.26 (t, 4H). LRMS (Electrospray,positive): Da/e 223.3 (m+1).

EXAMPLE 59 2-Hydroxy-4-morpholin-4-yl-N-benzyl-benzamide

[0442] Prepared via the Weinreb amidation procedure of Example 56.

[0443]¹H NMR (CDCl₃, 400 MHz): δ12.55 (d, 1H), 7.39-7.27 (m, 511), 7.20(d, 1H), 6.38 (d, 1H), 6.34 (dd, 1H), 6.31 (br s, 1H), 4.62 (d, 2H),3.82 (t, 4H), 3.24 (t, 4H). LRMS (Electrospray, negative): Da/e 311.4(m−1).

EXAMPLE 60 2-Hydroxy-4-morpholin-4-yl-N-phenyl-benzamide

[0444] Prepared via the Weinreb amidation procedure of Example 56.

[0445]¹H NMR (d6-DMSO, 400 MHz): δ12.41 (s, 1H), 10.110 (s, 1H), 7.92(d, 1H), 7.67 (d, 2H), 7.34 (t, 2H), 7.10 (t, 1H), 6.56 (dd, 1H), 6.35(d, 1H), 3.70 (4H), 3.22 (t, 4H). LRMS (Eleetrospray, negative): Da/e297.3 (m−1).

EXAMPLE 61 N-Cyclopropyl-2-hydroxy-4-morpholin-4-yl-N-phenyl-benzamide

[0446] Prepared via the Weinreb amidation procedure of Example 56.

[0447]¹H NMR (CDCl₃, 400 MHz): δ12.55 (s, 1H), 7.13 (d, 1H),6.36-6.32(m, 2H), 6.18 (br s, 1H), 3.82 (t, 4H), 3.23 (t, 4H), 2.84 (c,1H), 0.92-0.83 (m, 2H), 0.65-0.60 (m, 2H). LRMS (Electrospray,negative): Da/e 261.4 (m−1).

EXAMPLE 62 2-Hydroxy-4-morpholin-4-yl-N-(2-methoxyethyl)-benzamide

[0448] Prepared via the Weinreb amidation procedure of Example 56.

[0449]¹H NMR (CDCl₃, 400 MHz): δ12.56 (s, 1H), 7.22 (br s, 1H),6.45-6.31 (m, 2H), 3.82 (t, 4H), 3.60 (t, 2H), 3.55 (t, 2H), 3.39 (d,3H), 3.24 (t, 4H). LRMS (Electrospray, negative): Da/e 279.4 (m−1).

EXAMPLE 63 2-Hydroxy-4-morpholin-4-yl-N-methoxy-N-methyl-benzamide

[0450] Prepared via the Weinreb amidation procedure of Example 56.

[0451]¹H NMR (CDCl₃, 400 MHz): δ12.06 (s, 1H), 7.95 (dd, 1H), 6.39-6.34(m, 2H), 3.83 (t, 4H), 3.66 (s, 3H), 3.37 (s, 3H), 3.27 (t, 4H). LRMS(Electrospray, negative): Da/e 265.3 (m−1).

EXAMPLE 642-Hydroxy-4-morpholin-4-yl-N-(3-dimethylaminopropyl)-benzamide

[0452] Prepared via the Weinreb amidation procedure of Example 56.

[0453]¹H NMR (CDCl₃, 400 MHz.): δ8.89 (s, 1H), 7.13 (d, 1H), 6.37-6.34(m, 2H), 3.82 (t, 4H), 3.52 (t, 2H), 3.22 (dd, 4H), 2.52 (t, 2H), 2.31(s, 6H), 1.76 (c, 2H). LRMS (Electrospray, negative): Da/e 306.3 (m−1).

EXAMPLE 65 2-Hydroxy-4-morpholin-4-yl-N-niethoxy-benzamide

[0454] Prepared via the Weinreb amidation procedure of Example 56.

[0455]¹H NMR (CDCl₃, 400 MHz): δ11.80 (s, 1H), 8.82 (s, 1H), 7.18 (d,1H), 6.35 (br s, 1H), 6.32 (d, 1H), 3.85 (s, 3H), 3.82 (t, 4H), 3.24 (t,4H). LRMS (Electrospray, negative): Da/e 251.0 (m−1).

EXAMPLe 662-Hydroxy-4-morpholin-4-yl-N-(2-methanesulfonylethyl)-benzamide

[0456] Prepared via the Weinreb amidation procedure of Example 56.

[0457]¹H NMR (CDCl₃, 400 MHz): δ12.28 (s, 1H), 7.25 (d, 1H), 6.98 (t,1H), 6.37 (d, 1H) 6.34 (t, 1H), 3.96 (q, 2H), 3.82 (t, 4H), 3.33 (t,2H), 3.24 (t, 4H), 2.99 (s, 3H). LRMS (Electrospray, negative): Da/e327.1 (m−1).

EXAMPLE 67 2-Hydroxy-4-morpholin-4-yl-N-pyridin-3-yl-benzamide

[0458] Prepared via the Weinreb amidation procedure of Example 56.

[0459]¹H NMR (d6-DMSO, 400 MHz): δ12.21 (s, 1H), 10.24 (s, 1H), 8.83 (s,1H), 8.30 (d, 1H), 8.09 (d, 1H), 7.89 (d, 1H), 7.37 (dd, 1H), 6.58 (d,1H), 6.35 (s 1H), 3.70 (d, 4H), 3.23 (d, 4H). LRMS (Electrospray,negative): Da/e 298.4 (m−1).

EXAMPLE 68 2-Hydroxy-4-morpholin-4-yl-N-pyridin-4-yl-benzamide

[0460] Prepared via the Weinreb amidation procedure of Example 56.

[0461]¹H NMR (d6-Acetone, 400 MHz): δ9.71 (s, 1H), 8.48 (dd, 1H), 7.85(d, 1H), 7.75 (dd, 2H), 6.56 (dd, 1H), 6.38 (d, 1H), 3.76 (t, 4H), 3.30(t, 4H). LRMS (Electrospray, negative): Da/e 298.5 (m−1). LRMS(Electrospray, positive): Da/e 300.3 (m+1).

EXAMPLE 69 2-Hydroxy-4-morpholin-4-yl-N-thiazol-2-yl-benzamide

[0462] Prepared via the Weinreb amidation procedure of Example 56.

[0463]¹H NMR (d6-DMSO, 400 MHz): δ7.93 (d, 1H), 7.51 (d, 1H), 7.23 (brs, 1H), 6.60 (dd, 1H), 6.39 (d, 1H), 3.72 (t, 4H), 3.25 (t, 4H). LRMS(Electrospray, negative): Da/e 304.1 (m−1).

EXAMPLE 70 2-Hydroxy-4-morpholin-4-yl-N-(1,4-thiazin-2-yl)-benzamide

[0464] Prepared via the Weinreb amidation procedure of Example 56.

[0465]¹H NMR (d6-Acetone, 400 MHz), δ12.33 (s, 1H), 10.88 (s, 1H),7.52-7.37 (m, 6H), 6.48-6.34 (m, 2H), 5.00 (s, 2H), 3.75 (t, 4H), 3.25(t, 4H). LRMS (Electrospray, negative): Da/e 327.1 (m−1).

EXAMPLE 71 2,N-Dihydroxy-4-morpholin-4-yl-benzamide

[0466] Hydrogenation Procedure

[0467] To a stirred solution of o-benzyl hydroxamate (80 mg; 0.24 mmol)in dry MeOH (3 mL) and dry tetrahydrofuiran (3 mL) was carefully added10% palladium on carbon (6 mg; cat.) under nitrogen atmosphere at roomtemperature. The resulting mixture was purged on nitrogen and placedunder an atmosphere of hydrogen (balloon pressure) and allowed to stirfor 1.5 hours. The reaction was then carefully filtered through GF/Fpaper with suction (nitrogen blanket) and washed with CH₂Cl₂. Thefiltrate then was concentrated in vacuo and the residue recrystallizedfrom acetone/toluene to provide the hydroxamic acid as an off-whitesolid (38 mg; 66%).

[0468]¹H NMR (d6-Acetone, 400 MHz): δ12.28 (br s, 1H), 10.69 (br s, 1H),8.29 (br s, 1H), 7.54 (dd, 1H), 6.48 (dt, 1H), 6.34 (t, 1H), 3.76 (t,4H), 3.24 (t, 4H). LRMS (Electrospray, negative): Da/e 237.0 (m−1).

EXAMPLE 72 2-Hydroxy-4-morpholin-4-yl-N-(4-pyridylmethyl)-benzamide

[0469] Prepared via the Weinreb amidation procedure of Example 56.

[0470]¹NMR (d4-MeOH, 400 MHz): δ8.46 (dd, 2H), 7.65 (d, 1H), 7.38 (dd,2H), 6.50 (dd, 1H), 6.35 (d, 1H), 4.88 (s, 2H), 3.79 (t, 4H), 3.23 (t,4H). LRMS (Electrospray, positive): Da/e 314.4. (m+1). LRMS(Electrospray, negative): Da/e 312.3 (m−1).

EXAMPLE 731-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-(4-phenylpiperizin-1-yl)-methanone

[0471] Prepared via the Weinreb amidation procedure of Example 56.

[0472]¹H NMR (d6-DMSO, 400 MHz): δ9.80 (s, 1H), 7.24 (dd, 2H), 7.08 (d,1H), 6.97 (d, 2H), 6.82 (t, 1H), 6.49 (dd, 1H), 6.38 (d, 1H), 3.74 (t,4H), 3.59 (br s, 4H), 3.17-3.10 (m, 8H). LRMS (Electrospray, positive):Dale 368.3 (m+1). LRMS (Electrospray, negative): Dale 366.3 (m−1).

Example 74 2-Hydroxy-4-morpholin-4-yl-benzoic acid

[0473] Hydrolysis Procedure

[0474] To a stirred solution of 2-hydroxy-4-morpholin-4-yl-benzoic acidmethyl ester (4.6 g; 19.4 mmol) in MeOH (120 mL) was added water (60 mL)and lithium hydroxide hydrate (4.08 g; 97 mmol). The resulting mixturewas heated to 80° C. for 15 hours. The resulting mixture then was washedwith EtOAc (3×60 mL), and cooled to 0° C. and acidified with 2 N aqueoushydrochloric acid to pH 4. The resulting precipitate was collected on aBuchner funnel with suction , washed with water, and air dried toprovide the acid (4.16 g; 96%).

[0475]¹H NMR (d6-DMSO, 400 MHz): δ13.21 (br s, 1H), 11.37 (br s, 1H),7.56 (d, 1H), 6.48 (dd, 1H), 6.32 (d, 1H), 3.68 (t, 4H), 3.23 (t, 4H).

Example 75N-(Carboxymethyl-2-hydroxy-4-morpholin-4-yl-phenyl)-carboxamide methylester

[0476] EDC Coupling Procedure

[0477] To a stirred mixture of 2-hydroxy-4-morpholin-4-ylbenzoic acid(300 mg; 1.34 mmol) in CH₂Cl₂ (4 mL) and tetrahydrofuran (4 mL) wasadded glycine methyl ester hydrochloride (676 mg; 5.36 mmol),1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide hydrochloride (258 mg;1.34 mmol), and Hunig's base (0.937 mL; 5.36 mmol) at room temperatureunder a nitrogen atmosphere. The resulting solution was allowed to stirat room temperature for 24 hours, then concentrated at reduced pressureand re-dissolved in EtOAc (30 mL). The resulting solution was washedwith water (3×10 mL), 10% aqueous citric acid, 5% aqueous NaHCO₃ andbrine, then dried (Na₂SO₄), filtered and concentrated in vacuo. Theresidue was recrystallized from EtOAc/hexanes to provide the amide as awhite solid (79%).

[0478]¹H NMR (d6-Actone, 400 MHz): δ12.62 (s, 1H), 8.18 (br s, 1H), 7.66(d, 1H), 6.52 (dd, 1H), 6.34 (d, 1H), 4.13 (d, 2H), 3.78 (t, 4H), 3.71(s, 3H), 3.27 (t, 4H). LRMS (Electrospray, negative): Da/e 293.1 (m−1).

Example 76 N-Carboxymethyl-2-hydroxy-4-morpholin-4-yl-phenyl-carboxamide

[0479] Prepared via the hydrolysis procedure of Example 74.

[0480]¹H NMR (d4-MeOH, 400 MHz): δ7.63 (d, 1H), 6.50 (dd, 1H), 6.34 (d,1H),4.88 (s, 2H), 3.80 (t, 4H), 3.23 (t, 4H). LRMS (Electrospray,negative): Dale 279.0 (m−1).

Example 77 2-Hydroxy-4-morpholin-4-yl-thiobenzamide

[0481] To a stirred solution of 2-hydroxy-4-morpholin-4-yl-benzamide(102 mg; 0.46 mmol) and t-butyldimethylsilyl chloride (73 mg; 0.48 mmol)in dry tetrahydrofuran (4 mL) was added Hunig's base (0.084 mL; 0.48mmol) via syringe at room temperature under a nitrogen atmosphere. Afterstirring at room temperature for 4 hours, Lawesson's reagent was added(117 mg; 0.28 mmol) in one portion and the resulting solution wasstirred for 16 hours. Benzyl trimethyl ammonium fluoride (1.84 mmol)then was added and stirring was continued for 2 hours longer. Thereaction was concentrated at reduced pressure, and the residuere-dissolved in EtOAc (10 mL) and washed with water, 10% citric acid, 5%NaHCO₃, and brine then dried (Na₂SO₄), filtered and concentrated invacuo. The residue was then recrystallized from EtOAc/hexanes to providethe thioamide as a yellow solid (31 mg; 28%).

[0482]¹H NMR (d6-Acetone, 400 MHz): δ12.65 (br s, 1H), 8.73 (br s, 1H),8.50 (br s, 1H), 7.72 (d, 1H), 6.51 (dd, 1H), 6.35 (d, 1H), 3.77 (t,4H), 3.30 (t, 4H). LRMS (Electrospray, positive): Dale 239.2 (m+1).

Example 782-(4-Ethylphenyl)-4-imino-7-morpholin-4-yl-benzo(e]-1,3,2-oxathiaphosphane-2-thione

[0483] To a stirred solution of 2-hydroxy-4-morpholin-4-yl-benzamide(0.53 g; 2.4 mmol) in dry toluene (20 mL) was added Lawesson's reagent(0.58 g; 1.44 mmol) and the resulting mixture was heated to 100° C. for0.5 h under a nitrogen atmosphere. The resulting yellow mixture wasallowed to cool to room temperature and was concentrated at reducedpressure and the residue purified via flash chromatography (1:1hexanes/EtOAc on silica gel) to provide the heterocycle as a brightyellow solid (0.79 g; 99%).

[0484]¹H NMR (CDCl₃, 400 Mz): δ8.3 5 (d, 1H), 8.21 (br d, 1H), 7.84 (dd,2H), 6.95 (dd, 2H), 6.66 (dd, 1H), 6.35 (d, 1H), 3.85 (s, 3H), 3.82 (t,4H), 3.33 (t, 4H). LRMS (Electrospray, positive): Dale 405.1 (m+1).

Example 79 1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-phenyl-methanone

[0485] Ketone Procedure

[0486] To a stirred, cooled (−78° C.) solution of bromobenzene (0.475mL; 4.5 mmol) in dry tetrahydrofuran (10 mL) was added n-butyllithium(1.8 mL of 2.5 M in hexanes; 4.5 mmol) via syringe under nitrogenatmosphere. After stirring for 0.5 h at −78° C., the resulting solutionwas treated with a solution of theN-methyl-N-methoxy-(2-hydroxy-4-morpholin-4ylphenyl)-carboxamide (0.30g; 1.13 mmol),in dry tetrahydrofuran (2 mL) via carinula. After stirringat −78° C. for 3 hours, the reaction was quenched with the addition of10% aqueous citric acid. After warming to room temperature, thetetrahydrofuran was removed at reduced pressure and the residueextracted with EtOAc (3×15 mL). Combined organic layers were washed withwater and brine, then dried (NaSO₄), filtered and concentrated in vacuo.Purification of the residue via flash chromatography (10:1 hexanes/EtOAcon silica gel) provided the ketone as a light yellow crystalline solid(0.213 g; 70%).

[0487]¹H NMR (CDCl₃, 400 MHz): δ12.74 (s, 1H), 7.62 (d, 211), 7.55-7.43(m, 4H), 6.37 (d, 1H), 6.33 (dd, 1H), 3.82 (t, 4H), 3.35 (t, 4H). ¹³CNMR (CDCl₃, 100 MHz): δ199.0, 166.1, 156.6, 138.8, 135.4, 131.3, 129.0,128.4, 111.3, 105.3, 100.6, 66.7, 47.2.

Example 801-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-(4-trifluoromethylphenyl)-methanone

[0488] Prepared via the ketone procedure of Example 79.

[0489]¹H NMR (CDCl₃, 400 MHz): δ12.56 (s, 1H), 7.73 (q, 4H), 7.32 (d,1H), 6.37 (d, 1H), 6.33 (dd, 1H), 3.83 (t, 4H), 3.37 (t, 4H). ¹³C NMR(CDCl₃, 100 MHz): δ197.2, 166.1, 156.8, 141.9, 135.0, 128.0, 125.4 (q),110.9, 105.5, 100.4, 66.7, 47.1. LRMS (Electrospray, negative): Da/e350.3 (m−1).

EXAMPLE 81 1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-(o-tolyl)-methanone

[0490] Prepared via the ketone procedure of Example 79.

[0491]¹H NMR (CDCl₃, 400 MHz): δ12.79 (s, 1H), 7.38-7.31 (c, 1H),7.28-7.22 (m, 3H), 7.10 (d, 1H), 6.35 (d, 1H), 6.26 (dd, 1H), 3.81 (t,4H), 3.34 (t, 4H), 2.29 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz): δ201.1,165.8, 156.8, 138.5, 135.4, 135.3, 130.8, 129.7, 127.4, 125.4, 112.2,105.4, 100.3, 66.7, 47.2, 30.1, 19.9. LRMS (Electrospray, positive):Da/e 298.2 (m+1).

EXAMPLE 821-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-(4-methoxyphenyl)-methanone

[0492] Prepared via the ketone procedure of Example 79.

[0493]¹H NMR (CDCl₃, 400 MHz): δ12.75 (s, 1H), 7.65 (d, 2H), 7.50 (d,1H), 6.98 (d, 2H), 6.38 (d, 1H), 6.35 (dd, 1H), 3,88 (s, 3H1), 3.83 (t,4H), 3.35 (t, 4H). ¹³C NMR (CDCl₃, 100 MHz): δ1979, 165.8, 162.4, 156.4,135.2, 131.4, 131.2, 113.8, 111.5, 105.2, 100.8, 66.8, 55.8, 47.3. LRMS(Electrospray, negative): Da/e 312.2 (m−1).

EXAMPLE 831-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-pyridin-3-yl-methanone

[0494] Prepared via the ketone procedure of Example 79.

[0495]¹H NMR (CDCl₃, 400 MHz): δ12.56 (s, 1H), 8.86 (dd, 1H), 8.77 (dd,1H), 7.95 (dt, 1H), 7.44 (ddd, 1H), 7.37 (d, 1H), 6.37 (t, 1H), 6.34 (d,1H), 3.84 (t, 4H), 3.38 (t, 4H). ¹³C NMR (CDCl₃, 100 MHz): δ196.0,166.1, 15 6.8, 151.9, 149.6, 136.4, 134.9, 134.4, 123.5, 111.1, 105.6,100.4, 66.7, 47.1. LRMS (Electrospray, negative): Da/e 283.7 (m−1). LRMS(Electrospray, positive): Da/e 285.3 (m+1).

EXAMPLE 84 1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-pentan-1-one

[0496] Prepared via the ketone procedure of Example 79.

[0497]¹H NMR (CDCl₃, 400 MHz): δ12.88 (s, 1H), 7.60 (d, 1H), 6.37 (dd,1H), 6.28 (d, 1H), 3.82 (t, 4H), 3.31 (t, 4H), 2.85 (t, 2H), 1.70 (p,2H), 1.40 (h, 2H), 0.94 (t, 3H). ¹³C NMR (CDCl₃, 100 MHz): δ204.4,165.1, 156,5, 131.8, 111.8, 105.5, 100.8, 66.8, 47.3, 37.7, 27.6, 22.9,14.3. LRMS (Electrospray, negative): Da/e 262.3 (m−1),

EXAMPLE 85 1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-2-phenyl-ethanone

[0498] Prepared via the ketone procedure of Example 79.

[0499]¹H NMR (CDCl₃, 400 MHz): δ12.72 (s, 1H), 7.69 (d, 1H), 7.35-7.24(m, 5H), 6.37 (dd, 1H), 6.27 (d, 1H), 4.17 (s, 2H), 3.81 (t, 4H), 3.31(t, 4H). ¹³C NMR (CDCl₃, 100 MHz). δ200.9, 165.5, 156,6, 135.1, 132.3,129.5, 128.9, 127.1, 111.4, 105.7, 100.6, 66.7, 47.2, 44.9.

EXAMPLE 861-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-thiophen-2-yl-methanone

[0500] Prepared via the ketone procedure of Example 79.

[0501]¹H NMR (CDCl₃, 400 MHz): δ12.49 (s, 1H), 7.86 (d, 1H), 7.66 (d,2H), 7.17 (t, 1H), 6.42 (dd, 1H), 6.37 (d, 1H), 3.84 (t, 4H), 3.37 (t,4H). ¹³C NMR (CDCl₃, 100 MHz): δ188.7, 165.6, 156.4, 142.7, 133.8,132.9, 132.4, 127.7, 111.3, 105.6, 100.8, 66.8, 47.2. LRMS(Electrospray, positive): Da/e 290.2 (m+1).

EXAMPLE 871-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1,3-thiazol-2-yl-methanone

[0502] Prepared via the ketone procedure of Example 79.

[0503]¹H NMR (CDCl₃, 400 MHz): δ12.74 (s, 1H), 8.97 (d, 1H), 8.19 (q,2H), 6.64 (dd, 1H), 6.35 (d, 1H), 3.69 (t, 4H), 3.41 (t, 4H). ¹³C NMR(d6-DMSO, 100 MHz): δ182.3, 168.7, 167.0, 157.3, 145.7, 135.5, 127.9,108.9, 107.0, 99.4, 66.5, 46.9. LRMS (Electrospray, positive): Da/e291.2 (m+1).

EXAMPLE 881-(3-Chlorophenyl)-1-(2-hydroxy-4-morpholin-4-yl-phenyl)-methanone

[0504] Prepared via the ketone procedure of Example 79.

[0505]¹H NMR (CDCl₃, 400 MHz): δ12.56 (s, 1H), 7.59 (t, 1H), 7.52-7.48(m, 2H), 7.42 (d, 1H), 7.37 (d, 1H), 6.36 (t, 1H), 6.33 (d, 1H), 3.83(t, 4H), 3.36 (t, 4H), ¹³CNMR(CDCl₃, 100 MHz): δ197.1, 166.1, 156.8,140.4, 135.1, 134.6, 131.3, 129.8, 128.9, 127.0, 111.0, 105.5, 100.5,66.7, 47.1. LRMS (Electrospray, positive): Da/e 318.3 (m+1).

EXAMPLE 89 2-Chloro-1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone

[0506] Sugasawa Procedure

[0507] An oven-dried, 500-mL 3-necked round-bottom flask equipped withmechanical stirrer, condenser with a nitrogen inlet and dropping funnelwith rubber septum was charged with 5.0 g (27.8 mmol) of3-morpholinylphenol, 2.1 mL (33.8 mmol) chloroacetonitrile, and 150 mLof dichloroethane, and the mixture chilled in an ice bath. The droppingfunnel was charged with 100 mL (100 mmol) of 1.0 M boron trichloride inCH₂Cl₂ and this was added dropwise with vigorous stirring. When theaddition was completed 1.9 g (14 mmol) of aluminum trichloride was addedin one portion. The reaction then was heated at 60° C. for 18 h. Thereaction then was chilled (0° C.). The dropping funnel was charged with100 mL of 2 N aqueous hydrochloric acid, which was added dropwiseforming a solid. When the addition was complete, the reaction was heatedat 60° C. for 1 h which dissolved most of the solids. The reaction wascooled, and the organic layer separated and washed with 2 N HCl (2×40mL), water (40 mL), and brine (40 mL). The organic layer then was dried(MgSO₄), filtered and concentrated in vacuo to give 1.8 g (25.5%) of thedesired chloroketone as a yellowish green solid.

[0508]¹H NMR (CDCl₃, 400 MHz): δ12.16 (s, 1H), 7.53 m (d, 1H), 6.39 (dd,1H), 6.29 (d, 1H), 4.56 (d, 2H), 3.82 (t, 4H), 3.36 (t, 4H).

EXAMPLE 901-(2-Hydroxy-4-morpholin-4-yl-phenyl)-2-morpholin-4-yl-ethanone

[0509] Amination Procedure

[0510] To a stirred solution of 2-chloro-1-(2-hydroxy4-morpholin-4-ylphenyl)-ethanone (95 mg; 0.37 mmol) in dry acetonitrile (3 mL) was addedpowdered K₂CO₃ (77 mg; 0.56 mmol) and morpholine (0.034 mL; 0.39 mmol)at room temperature under a nitrogen atmosphere. The resulting mixturewas heated to reflux for 0.5 hour, then allowed to cool to roomtemperature. The reaction was diluted with CH₂Cl₂ (50 mL), filtered, andconcentrated in vacuo.

[0511] The residue was purified by radial chromatography (2 mmchromatotron plate with 2% MeOH in CH₂Cl₂) to provide the amine as aclear, colorless oil (87 mg; 77%).

[0512]¹H NMR (CDCl₃, 400 MHz): δ12.68 (br s, 1H), 7.72 (d, 1H), 6.33(dd, 1H), 6.22 (d, 1H), 3.77 (t, 4H), 3.71 (t, 4H), 3.62 (s, 2H), 3.28(t, 4H), 2.55 (t, 4H). LRMS (Electrospray, positive): Da/e 307.4 (m+1).

EXAMPLE 91 1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-2imidazol-1-yl-ethanone

[0513] Prepared via the amination procedure of Example 90.

[0514]¹H NMR (CDCl₃, 400 MHz): δ12.05 (br s, 1H), 7.55-7.50 (m, 2H),7.14 (s, 1H), 6.95 (s, 1H), 6.42 (dd, 1H), 6.29 (d, 1H), 5.28 (s, 2H),3.83 (t, 4H), 3.36 (t, 4H). LRMS (Electrospray, positive): Da/e 288.3(m+1). LRMS (Electrospray, negative): Da/e 286.3 (m−1).

EXAMPLE 921-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-pyrrolidin-1-yl-methanone

[0515] Prepared via the amination procedure of Example 90.

[0516]¹H NMR (CDCl₃, 400 MHz): δ7.77 (d, 1H), 6.38 (dd, 1H), 6.26 (d,1H), 3.82 (t, 4H), 3.77 (s, 2H), 3.30 (t, 4H), 2.71 (br s, 4H),1.89-1.82 (m, 4H). LRMS (Electrospray, positive): Da/e 291.3 (m+1).

EXAMPLE 931-(2-Hydroxy-4-morpholin-4-yl-phenyl)-1-(4-methyl-piperazin-1-yl)-methanone

[0517] Prepared via the amination procedure of Example 90.

[0518]¹H NMR (CDCl₃, 400 MHz): δ7.76 (d, 1H), 6.35 (dd, 1H), 6.25 (d,1H), 3.79 (t, 4H), 3.62 (s, 2H), 3.29 (t, 4H), 2.62-2.29 (m, 8H), 2.28(s, 3H). LRMS (Electrospray, positive): Da/e 320.4 (m+1). LRMS(Electrospray, negative): Da/e 318.4 (m−1).

EXAMPLE 94 2-Hydroxy-4-morpholin-4-yl-phenyl-1-piperidin-1-yl-methanone

[0519] Prepared via the amination procedure of Example 90.

[0520]¹H NMR (CDCl₃, 400 MHz): δ7.83 (d, 1H), 6.37 (dd, 1H), 6.25 (d,1H), 3.80 (t, 4H), 3.53 (s, 2H), 3.29 (t, 4H), 2.55 (br s, 4H),1.70-1.62 (m, 4H), 1.47 (br s, 2H). LRMS (Electrospray, positive): Da/e305.4 (m+1).

EXAMPLE 952-(Benzyl-methyl-amino)-1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone

[0521] Prepared via the amination procedure of Example 90.

[0522]¹H NMR (CDCl₃, 400 MHz): δ7.75 (d, 1H), 7.37-7.25 (m, 5H), 6.34(dd, 1H), 6.26 (d, 1H), 3.81 (t, 4H), 3.64 (d, 2H), 3.31 (t, 4H), 2.33(s, 3H). LRMS (Electrospray, positive): Da/e 341.4 (m+1).

EXAMPLE 96 2-Acetylthio-1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone

[0523] Prepared via the amination procedure of Example 90 with potassiumthioacetate:

[0524]¹H NMR (CDCl₃, 400 MHz): δ12.30 (s, 1H), 7.64 (d, 1H), 6.39 (dd,1H), 6.26 (d, 1H), 4.29 (s, 2H), 3.82 (t, 4H), 3.34 (t, 4H), 2.40 (s,3H). ¹³C NMR (CDCl₃, 100 MHz): δ195.6, 194.1, 165.2, 156.8, 131.9,110.5, 100.3, 66.7, 47.1, 35.6, 30.5. LRMS (Electrospray, positive):Da/e 296.1 (m+1).

[0525] Example 97

1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-2-mercapto-ethanone

[0526] To a stirred solution of thioester (74.1 mg; 0.25 mmol) in dryMeOH (2 mL) was added sodium methoxide (5 mg; 0.1 mmol) at roomtemperature under a nitrogen atmosphere. The resulting mixture wasallowed to stir for 22 hours, then concentrated at reduced pressure andredissolved in CH₂Cl₂ (10 mL). This solution was washed with 1 N aqueoushydrochloric acid (2×3 mL), water and brine then dried (MgSO₄), filteredand concentrated in vacuo. The residue was purified via radialchromatography (1 mm chromatotron plate with 20% EtOAc in hexanes) toprovide the free thiol as a pale yellow solid (10.1 mg; 16%).

[0527]¹H NMR (CDCl₃, 400 MHz): δ12.46 (s, 1H), 7.53 (d, 1H), 6.37 (dd,1H), 6.26 (d, 1H), 4.04 (s, 2H), 3.82 (t, 4H), 3.34 (t, 4H). LRMS(Electrospray, negative): Da/e 252.0 (m−1).

EXAMPLE 98 6-Morpholin-4-yl-2-hydrobenzo(b]furan-3-one

[0528] Prepared via the amination procedure of Example 90 via omissionof amine.

[0529]¹H NMR (CDCl₃, 400 MHz): δ7.50 (d, 1H), 6.59 (dd, 1H), 6.36 (d,1H), 4.57 (s, 2H), 3.83 (t, 4H), 3.35 (t, 4H). ¹³C NMR (CDCl₃, 100 MHz):δ196.9, 176.5, 158.6, 125.2, 112.2, 109.7, 95.6, 75.7, 66.7, 47.7.

EXAMPLE 994-(2-Methyl-4-morpholin-4-yl-phenyl)-2-(3-pyridyl)-1,3-thiazole

[0530] Thiazole Cyclization Procedure

[0531] A stirred solution of chioro-ketone (76 mg; 0.30 mmol) and3-thioamide-pyridine (41 mg; 0.30 mmol) in ethanol (2 mL) was heated toreflux for 5 hours under a nitrogen atmosphere. The reaction was allowedto cool to room temperature, then concentrated in vacuo. The dark oilwas purified by radial chromatography (2 mm chromatotron plate with 5%MeOH in CH₂Cl₂) to provide the heterocycle as a yellow oil (27 mg; 27%).

[0532]¹H NMR (CDCl₃, 400 MHz): δ11.53 (br s, 1H), 9.16 (d, 1H), 8.69(dd, 1H), 8.21 (dt, 1H), 7.51 (d, 1H), 7.45-7.40 (m, 2H), 6.51 (t, 1H),6.49 (d, 1H), 3.86 (t, 4H), 3.22 (t, 4H). LRMS (Electrospray, positive):Da/e 340.3 (m+1). LRMS (Electrospray, negative): Da/e 338.3 (m−1).

EXAMPLE 100 5-Morpholin-4-yl-2-(2-phenylamino-1,3-thiazol-4-yl)-phenol

[0533] Prepared via the thiazole cyclization procedure of Example 99.

[0534]¹H NMR (d6-DMSO, 400 MHz): δ11.02 (s, 1H), 10.34 (s, 1H), 7.68 (d,1H), 7.49 (d, 2H), 7.34 (t, 2H), 7.14 (s, 1H), 6.99 (t, 1H), 6.50 (dd,1H), 6.37 (d, 1H), 3.71 (t, 4H), 3.09 (t, 4H). LRMS (Electrospray,negative): Da/e 352.2 (m−1).

EXAMPLE 101 3-Methoxy-1-morpholin-4-yl-benzene

[0535] To a stirred mixture of 3-morpholinyl-phenol (3.8 g; 21.2 mmol)and powdered K₂CO₃ (4.4 g; 31.8 mmol) in dry DMF (15 mL) was addediodomethane (1.45 mL; 23.3 mmol) via syringe at room temperature undernitrogen atmosphere. The resulting mixture was allowed to stir at roomtemperature for 22 hours. The reaction was diluted with ether (150 mL)and washed successively with water (2×40 mL) and brine (40 mL), thendried (MgSO₄), filtered and concentrated in vacuo to provide the desiredether as a yellow oil (2.51 g; 61%).

[0536]¹H NMR (CDCl₃, 400 MHz): δ7.19 (t, 1H), 6.53 (ddd, 1H), 6.45 (d,1H), 3.85 (t, 4H), 3.80 (s, 3H), 3.15 (t, 4H). LRMS (Electrospray,positive): Da/e 194.1 (m+1).

EXAMPLE 102 4-Methoxy-2-morpholin-4-yl-benzenesulfonic acid

[0537] To a cooled (0° C.), stirred portion of1-methoxy-3-morpholin-4-yl-benzene (1.08 g; 5.6 mmol) was added neat tochlorosulfonic acid (1.8 mL; 28 mmol) via pipette over 2 minutes undercalcium sulfate-dried atmosphere. The resulting thick, red solution wasallowed to warm to room temperature and stirred for 5 hours. Theresulting solution was poured onto ice, then extracted with CH₂Cl₂ (3×50mL). Combined organic layers then were washed with brine, dried (MgSO₄),filtered and concentrated in vacuo to give the acid as a light greensolid (0.31 g; 20%).

[0538]¹H NMR (CDCl₃, 400 MHz): δ8.14 (d, 1H), 7.08 (dd, 1H), 6.85 (t,1H), 4.23 (dd, 1H), 4.11-4.01 (m, 2H), 3.90 (d, 3H), 3.86 (t, 1H), 3.67(d, 1H), 3.57-3.50 (m, 2H), 3.37 (t, 1H). LRMS (Electrospray, negative):Da/e 272.0 (m−1).

EXAMPLE 103 4-Methoxy-2-morpholin-4-yl-benzenesulfonyl chloride

[0539] A mixture of 4-methoxy-2-morpholin-4-yl-benzenesulfonyl chloride(0.31 g; 1.14 mmol) and thionyl chloride (25 mL) was refluxed under acalcium sulfate-dried atmosphere for 4 hours. Excess thionyl chloridewas distilled from the residue, which was dried in vacuo to provide thecrude sulfonyl chloride as a brown solid (0.30 g; 90%).

[0540]¹H NMR (CDCl₃, 400 MHz): δ7.98 (d, 1H), 6.86 (d, 1H), 6.77 (dd,1H), 3.90-3.86 (m, 7H), 3.07-3.04 (m, 4H).

EXAMPLE 104 4-Methoxy-2-morpholin-4-yl-N-methyl-benzenesulfonamide

[0541] Sulfonylation Procedure

[0542] To a stirred solution of4-methoxy-2-morpholin-4-yl-benzenesulfonyl chloride (37 mg; 0. 13 mmol)in dry CH₂Cl₂ (1 mL) was added methylamine (0.16 mL of 2.0 M solution intetrahydrofuran; 0.32 mmol) via syringe at room temperature under anitrogen atmosphere. After stirring for 15 hours, the reaction wasdiluted with CH₂Cl₂ (25 mL), washed with water, saturated aqueous NaHCO₃and brine, then dried (MgSO₄), filtered, and concentrated in vacuo. Theresidue was purified via HPLC (Biotage with 2:1 hexanes/EtOAc) toprovide the sulfonamide as a white solid (12.5 mg; 34%).

[0543]¹H NMR (CDCl₃, 400 MHz): δ7.95 (d, 1H), 6.87 (d, 1H), 6.80 (dd,1H), 5.68 (br d, 1H), 3.87 (s, 7H), 3.03 (t, 4H), 2.47 (d, 3H). ¹³C NMR(CDCl₃, 100 MHz): 6 164.1, 152.3, 132.9, 126.2, 110.3, 109.9, 67.9,56.0, 54.5, 30.1. LRMS (Electrospray, positive): Da/e 287.2 (m+1). LRMS(Electrospray, negative): Da/e 285.4 (m−1).

EXAMPLE 105 4-Methoxy-2-morpholin-4-yl-N-benzyl-benzenesulfonamide

[0544] Prepared via the sulfonylation procedure of Example 104.

[0545]¹H NMR (CDCl₃, 400 MHz): δ8.00 (dd, 1H), 7.23-7.19 (m, 3H), 7.02(dd, 2H), 6.82 (dd, 1H), 6.73 (d, 1H), 6.08 (t, 1H), 3.98 (d, 2H), 3.87(d, 3H), 3.64 (br s, 4H), 2.77 (t, 4H). ¹³C NMR (CDCl₃, 100 MHz):δ164.1, 152.3, 136.7, 132.2, 128.9, 128.3, 128.2, 127.9, 110.3, 109.7,67.7, 56.0, 54.2, 48.6. LRMS (Electrospray, positive): Da/e 363.3 (m+1).LRMS (Electrospray, negative): Da/e 361.7 (m−1).

EXAMPLE 1064-Methoxy-2-morpholin-4-yl-N-cyclopropylmethyl-benzenesulfonamide

[0546] Prepared via the sulfonylation procedure of Example 104.

[0547]¹H NMR (CDCl₃, 400 MHz): δ7.99 (dd, 1H), 6.87 (d, 1H), 6.80 (dd,1H), 6.12 (br s, 1H), 3.87-3.85 (m, 7H), 3.04 (t, 4H), 1.95 (c, 1H),0.64-0.57 (m, 2H), 0.52-0.48 (m, 2H). ¹³C NMR (CDCl₃, 100 MHz): δ164.2,152.4, 132.8, 127.5, 110.4, 109.9, 67.9, 56.0, 54.4, 25.2, 6.1. LRMS(Electrospray, negative): Da/e 311.7 (m−1).

EXAMPLE 107 N,N-Diethyl-(3-morpholin-4-yl-phenoxy)carboxamide

[0548] To a stirred mixture of 3-morpholinylphenol (0.34 g; 1.9 mmol)and powdered K₂CO₃ (0.576 g; 4.2 mmol) in dry acetonitrile (10 mL) wasadded diethyl carbamylchloride (0.26 mL; 2.0 mmol) via syringe at roomtemperature under nitrogen atmosphere. The resulting mixture was heatedto reflux for 24 hours, then allowed to cool to room temperature. Thereaction was diluted with EtOAc (60 mL), washed with water (2×20 mL) andbrine, then dried (MgSO₄), filtered, and concentrated in vacuo. Theresidue was purified via radial chromatography (4 mm chromatotron platewith 2:1 hexanes/EtOAc) to provide the carbamate as a clear, colorlessoil (0.39 g; 74%).

[0549]¹H NMR (CDCl₃, 400 MHz; mixture of rotomers): δ7.22 (dt, 1H), 6.72(ddd, 1H), 6.66 (t, 1H), 6.62 (ddd, 1H), 3.83 (dt, 4H), 3.40 (br q, 4H),3.15 (dt, 4H), 1.26-1.18 (m, 6H). LRMS (Electrospray, positive): Da/e279.1 (m+1).

EXAMPLE 108 N,N-Diethyl-(2-benzenesulfonyl-5-morpholin-4-yl-phenoxy)carboxamide

[0550] To a cooled (−78° C.), stirred solution of carbamate (95 mg; 0.34mmol) in dry tetrahydrofuran (2 mL) and tetramethylethylenediamine (0.4mL) was added sec-butyllithium (0.29 mL of a 1.3 M solution incyclohexane; 0.38 mmol) via syringe under a nitrogen atmosphere. Theresulting solution was allowed to stir at −78° C. for 0.6 hour, thenbenzenesulfonyl fluoride (0.043 mL; 0.36 mmol) was added via syringe.The resulting light yellow solution was allowed to stir at −78° C. for2.5 hours, then warmed to room temperature and stirred overnight. Thereaction was quenched with water, extracted with ether (3×20 mL).Combined organic layers were washed with brine, dried (MgSO₄), filtered,and concentrated in vacuo. The residue was purified via radialchromatography (2 mm chromatotron plate with 1:1 hexanes/EtOAc) toprovide the desired sulfone (49 mg; 67% based on recovered startingmaterial).

[0551]¹H NMR (CDCl₃, 400 MHz; mixture of rotomers): δ7.96 (dd, 1H), 7.79(dd, 2H), 7.50 (c, 1H), 7.42 (tt, 2H), 6.74 (dd, 1H), 6.57 (d, 1H), 3.79(t, 4H), 3.39 (q, 2H), 3.26 (t, 4H), 3.20 (q, 2H), 1.17 (t, 3H), 1.06(t, 3H). LRMS (Electrospray, positive): Da/e 419.3 (m+1).

EXAMPLE 109 2-Benzenesulfonyl-5-morpholin-4-yl-phenol

[0552] To a stirred solution of carbamate (31 mg; 0.07 mmol) in ethanol(1.5 mL) and water (0.3 mL) was added potassium hydroxide (118 mg; 2.1mmol). The resulting solution was heated to reflux for 5 hours, thenallowed to cool to room temperature. The reaction was neutralized withthe addition of aqueous hydrochloric acid (0.35 mL of 6.0 N solution;2.1 mmol), and the solution concentrated in vacuo. The residue waspurified via radial chromatography (2 mm chromatotron plate with 2:1hexanes/EtOAc) to provide the phenol as a clear semi-solid (15.6 mg;66%).

[0553]¹H NMR (CDCl₃, 400 MHz): δ9.16 (s, 1H), 7.89-7.87 (m, 2H),7.56-7.46 (m, 4H), 6.43 (dd, 1H), 6.32 (d, 1H), 3.78 (t, 4H), 3.24 (t,4H). ¹³C NMR (CDCl₃, 100 MHz): δ157.6, 156.7, 143.0, 133.3, 130.6,129.5, 126.5, 112.5, 107.3, 101.9, 66.7, 47.3. LRMS (Electrospray,negative): Da/e 318.0 (m−1).

EXAMPLE 110 3-Nitro-1 -morpholin-4-yl-benzene

[0554] Morpholine Ring Construction Procedure

[0555] A 2-L 3-necked roundbottom flask equipped with mechanicalstirrer, condenser with nitrogen inlet, and thermocouple was chargedwith 138.13 (1.0 mol) 3-nitrophenylamine, 152.0 g (1.1 mol) K₂CO₃, and 1L DMF. To this was added 129 mL (1.1 mol) of bis 2-chloroethylether and30 g (0.2 mol) NaI and the mixture heated at reflux for 72 h. Thereaction was allowed to cool to room temperature, then filtered, and thefiltrate concentrated in vacuo with water bath at 60° C. The resultingdark oil was treated with 500 mL MeOH and Darco, and heated on steambath for 1 h. The mixture then was filtered over GF/F paper and thefiltrate allowed to slowly cool to room temperature resulting in crystalformation. The mixture was placed in at 5° C. overnight then filteredcold, washing with MeOH. Upon drying recovered the desired arylmorpholine 54.9 g (26.3%) was recovered as orange needles.

[0556]¹H NMR (CDCl₃, 400 MHz): δ7.72-7.67 (m, 2H), 7.39 (t, 1H), 7.19(dd, 1H), 3.88 (t, 4H), 3.25 (t, 4H). LRMS (Electrospray, positive):Da/e 209.2 (m+1).

EXAMPLE 111 3-Morpholin-4-yl-phenylamine

[0557] Hydrogenation Procedure

[0558] A 500 mL roundbottom flask equipped with stir bar and 3-waystopcock charged with MeOH/tetrahydrofuran/water (200 mL of 2:1:1) waspurged thoroughly with nitrogen. To this was added 1.0 g of 10% Pd/Cfollowed by addition of 10 g (48.0 mmol) of 3-morpholinyl nitrobenzene.The mixture then was stirred under a hydrogen atmosphere for 4 days (1atm). The reaction was filtered over GF/F filter paper and the filtrateconcentrated in vacuo to remove MeOH and THF. The resulting solid wasrecovered by filtration, washed with water, air dried, then dried invacuo to give 7.3 g (83% yield) of the desired aniline as beigecrystals.

[0559]¹H NMR (CDClH, 400 MHz): δ7.06 (t, 1H), 6.35 (ddd, 1H), 6.26-6.23(m, 2H), 3.84 (t, 4H), 3.62 (br s, 2H), 3.12 (t, 4H). LRMS(Electrospray, positive): Da/e 179.2 (m+1).

EXAMPLE 112 1-(2-Amino-4-morpholin-4-yl-phenyl)-2-chloro-ethanone

[0560] Prepared via the Sugasawa procedure of Example 89.

[0561]¹H NMR (CDCl₃, 400 MHz): δ7.50 (d, 1H), 6.36 (br s, 2H), 6.22 (dd,1H), 5.94 (d, 1H), 4.57 (s, 2H), 3.81 (t, 4H), 3.27 (t, 4H). LRMS(Electrospray, positive): Da/e 255.3 (m+1).

EXAMPLE 113 2-Amino-4-morpholin-4-yl-N-benzyl-N-methyl-benzamide

[0562] Prepared via the amination procedure of Example 90.

[0563]¹H NMR (CDCl₃, 400 MHz): δ7.72 (d, 1H), 7.37-7.23 (m, 5H), 6.35(br s, 2H), 6.17 (dd, 1H), 5.93 (d, 1H), 3.81 (t, 4H), 3.64 (s, 2H),3.63 (s, 2H), 3.24 (t, 4H), 2.32 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz):δ197.3, 155.2, 152.9, 138.8, 133.3, 129.4, 128.4, 127.3, 110.6, 103.8,99.4, 66.9, 64.2, 62.6, 47.6, 43.1. LRMS (Electrospray, positive): Da/e340.1 (m+1).

EXAMPLE 1141-(2-Amino-4-morpholin-4-yl-phenyl)-1-pyrrolidin-1-yl-methanone

[0564] Prepared via the amination procedure of Example 90.

[0565]¹H NMR (CDCl₃, 400 MHz): δ7.68 (d, 1H), 6.34 (br s, 2H), 6.19 (dd,1H), 5.94 (d, 1H), 3.83 (s, 2H), 3.80 (t, 4H), 3.23 (t, 4H), 2.66 (br s,4H), 1.84-1.78 (m, 4H). ¹³CNMR(CDCl₃, 100MHz): δ196.3, 155.1, 152.7,132.5, 110.4, 104.0, 99.6, 66.9, 62.6, 54.7, 47.6, 24.0. LRMS(Electrospray, positive): Da/e 290.0 (m+1).

EXAMPLE 115 (2-Amino-4-morpholin-4-yl-phenyl)-1-piperidin-1-yl-methanone

[0566] Prepared via the amination procedure of Example 90.

[0567]¹H NMR (CDCl₃, 400 MHz): δ7.77 (d, 1H1), 6.34 (br s, 2H), 6.19(dd, 1H), 5.93 (d, 1H), 3.80 (t, 4H), 3.60 (s, 2H), 3.23 (t, 4H), 2.49(br s, 4H), 1.65-1.59 (m, 4H), 1.46-1.42 (m, 2H). ¹³C NMR (CDCl₃, 100MHz): δ196.5, 155.1, 152.8, 132.9, 110.8, 103.9, 99.5, 66.9, 65.9, 55.3,47.6, 26.2, 24.4. LRMS (Electrospray, positive): Da/e 304.0 (m+1).

EXAMPLE 116 2-Amino-4-fluoro-benzoic acid methyl ester

[0568] To a cooled (0° C.), stirred solution of 4-fluoro-2-aminobenzoicacid (2.68 g; 17.3 mmol) in dry tetrahydrofuran (60 mL) and MeOH (10 mL)was added a solution of trimethylsilyldiazomethane in hexanes (12 mL of2.0 N; 24 mmol) via syringe over 20 minutes under nitrogen atmosphere.The resulting solution was allowed to warm to room temperature andstirred for 2 hours then concentrated in vacuo to provide the ester alight yellow solid (˜100%).

[0569]¹H NMR (d6-DMSO, 400 MHz): δ7.76 (dd, 1H), 6.91 (br s, 2H), 6.53(dd, 1H), 6.34 (ddd, 1H), 3.78 (s, 3H).

EXAMPLE 117 4-Fluoro-2-(2,2,2-trifluoroacetylamino)-benzoic acid methylester

[0570] To a cooled (0° C.), stirred solution of methyl ester (0.525 g;3.1 mmol) in dry CH₂Cl₂ (20 mL) was added trifluoroacetic anhydride (0.5mL; 3.5 mmol) via syringe under a nitrogen atmosphere. The resultingsolution was allowed to stir for 20 minutes, then quenched with theaddition of MeOH (0.5 mL) and water (2 mL). The mixture was diluted withCH₂Cl₂ (20 mL) and washed with water (3×5 mL) and brine then dried(MgSO₄), filtered and concentrated in vacuo to provide the amide as alight yellow solid (89%).

[0571]¹H NMR (CDCl₃, 400 MHz): δ12.21 (br s, 1H), 8.42 (dd, 1H), 8.06(dd, 1H), 6.98 (ddd, 1H), 3.98 (s, 3H). LRMS (Electrospray, negative):Da/e 264.3 (m−1).

EXAMPLE 118 4-Morpholin-4-yl-2-(2,2,2-trifluoroacetylamino)-benzoic acidmethyl ester

[0572] Nucleophilic Addition of Morpholine Procedure

[0573] To a stirred solution of arylfluoride (0.727 g; 2.7 mmol) indimethylsulfoxide (20 mL) was added morpholine (0.95 mL; 10.9 mmol), andthe resulting solution was heated at 80° C. for 4 hours under a nitrogenatmosphere. After cooling to room temperature, the reaction was dilutedwith EtOAc (60 mL) and washed with I N aqueous hydrochloric acid. Theaqueous layer was back extracted with EtOAc (3×10 mL), and combinedorganic layers were washed with water (3×10 mL) and brine, then dried(MgSO₄), filtered, and concentrated in vacuo. The residue was purifiedvia flash chromatography (3:1 hexanes/EtOAc on silica gel) to providethe arylmorpholine as a white solid (629 mg; 70%).

[0574]¹H NMR (CDCl₃, 400 MHz): δ12.51 (s, 1H), 8.22 (d, 1H), 7.94 (d,1H), 6.62 (dd, 1H), 3.91 (s, 3H), 3.84 (t, 4H), 3.35 (t, 4H). LRMS(Electrospray, negative): Da/e 331.3 (m−1).

EXAMPLE 119 2-Amino-4-morpholin-4-yl-benzoic acid

[0575] Lithium Hydroxide Hydrolysis Procedure

[0576] To a stirred solution of ester-trifluoroamide (54 mg; 0.16 mmol)in tetrahydrofuran (1 mL), MeOH (0.5 mL) and water (0.5 mL) was addedlithium hydroxide monohydrate (30 mg; 0.71 mmol) at room temperature.The resulting solution was heated to 50° C. for 22 hours, then allowedto cool to room temperature. The mixture was neutralized with 1 Naqueous hydrochloric acid (0.7 mL; 0.70 mmol), then extracted with EtOAc(3×10 mL). Combined organic layers were washed with water and brine,then dried (MgSO₄), filtered and concentrated in vacuo. The residue waspurified via radial chromatography (2 mm chromatotron plate with 5% MeOHin CH₂Cl₂) to provide the acid as a light yellow solid.

[0577]¹H NMR (d6-DMSO, 400 MHz): δ7.52 (d, 1H), 6.19 (dd, 1H), 6.12 (d,1H), 3.68 (t, 4H), 3.11 (t, 4H). LRMS (Electrospray, negative): Da/e221.3 (m−1).

EXAMPLE 120 2-Methylsulfonylamino-4-morpholin-4-yl-benzoic acid

[0578] To a suspension of 2-amino-4-morpholin-4-ylbenzoic acid (230 mg;1.04 mmol) in CH₂Cl₂ (3 mL) was addedN-methyl-N-(trimethylsilyl)trifluoro-acetamide (0.4 mL; 2.1 mmol) viasyringe at room temperature under a nitrogen atmosphere. The resultingmixture was heated to reflux for 10 minutes, then allowed to cool toroom temperature. Methanesulfonyl chloride (1.2 eq.) and triethylamine(1.2 eq.) were added, and the resulting mixture allowed to stir for 2hours. The reaction was diluted with CH₂Cl₂ (30 mL) and washed withwater (2×10 mL) and brine, then dried (MgSO₄), filtered, andconcentrated in vacuo to provide the sulfonamide as a white solid (280mg; 90%).

[0579]¹H NMR (d6-DMSO, 400 MHz): δ10.88 (s, 1H), 7.80 (d, 1H), 6.92 (d,1H), 6.71 (dd, 1H), 3.71 (t, 4H), 3.25 (t, 4H), 3.16 (s, 3H). LRMS(Electrospray, negative): Da/e 299.0 (m−1).

EXAMPLE 1214-Morpholin-4-yl-2-(2,2,2-trifluoroacetylamino)-N-benzyl-benzamide

[0580] Prepared by the EDC Coupling procedure of Example 75.

[0581]¹H NMR (CDCl₃, 400 MHz): δ13.33 (s, 1H), 8.26 (d, 1H), 7.42-7.32(m, 6H), 6.60 (dd, 1H), 6.39 (br s, 1H), 4.63 (d, 2H), 3.84 (t, 4H),3.31 (t, 4H) LRMS (Electrospray, negative): Da/e 406.3 (m−1).

EXAMPLE 122N,N-Dimethyl-4-morpholin-4-yl-2-(2,2,2-trifluoroacetylamino)-benzamide

[0582] Prepared via the Weinreb amidation procedure of Example 56.

[0583]¹H NMR (CDCl₃, 400 MHz): δ11.26 (s, 1H), 7.99 (d, 1H), 7.27 (d,1H), 6.63 (dd, 1H), 3.84 (t, 4H), 3.28 (t, 4H), 3.11 (s, 6H). LRMS(Electrospray, positive): Da/e 346.3 (m+1). LRMS (Electrospray,negative): Da/e 344.3 (m−1).

EXAMPLE 123 2-Amino-4-morpholin-4-yl-N,N-dimethyl-benzamide

[0584] Prepared via the lithium hydroxide hydrolysis procedure ofExample 119 (without heat).

[0585]¹H NMR (CDCl₃, 400 MHz): δ7.00 (d, 1H), 6.23 (dd, 1H), 6.14 (d,1H), 4.58 (br s, 2H), 3.80 (t, 4H), 3.13 (t, 4H), 3.03 (s, 61H). LRMS(Electrospray, positive): Da/e 250.1 (m+1).

EXAMPLE 124N-Methyl-4-morpholin-4-yl-2-(2,2,2-trifluoroacetylamino0)-benzamide

[0586] Prepared via the Weinreb amidation procedure of Example 56.

[0587]¹H NMR (CDCl₃, 400 MHz): δ13.35 (s, 1H), 8.24 (d, 1H), 7.38 (d,1H), 6.61 (dd, 1H), 6.19 (br s, 1H), 3.84 (t, 4H), 3.31 (t, 4H), 3.00(d, 3H). LRMS (Electrospray, negative): Da/e 330.1 (m−1).

EXAMPLE 125 2-Amino-4-morpholin-4-yl-benzoic acid methyl ester

[0588] Prepared via the lithium hydroxide hydrolysis procedure ofExample 119 (without heating).

[0589]¹H NMR (CDCl₃, 400 MHz): δ7.74 (d, 1H), 6.23 (dd, 1H), 6.00 (d,1H), 5.71 (br s, 2H), 3.85-3.81 (mn, 711), 3.22 (t, 4H). LRMS(Electrospray, positive): Da/e 237.4 (m+1).

EXAMPLE 126 2-Acetylamino-4-morpholin-4-yl-benzole acid methyl ester

[0590] To a stirred solution of methyl 2-amino-4-morpholin-4-ylbenzoate(55 mg; 0.23 mmol) in dry CH₂Cl₂ (2 mL) was added acetic anhydride (0.05mL; 0.53 mmol) and pyridine (0.02 mL; 0.25 mmol) via syringe at roomtemperature under a nitrogen atmosphere. The resulting solution wasallowed to stir for 22 hours, then diluted with EtOAc (15 mL) and washedwith 0.5 N aqueous hydrochloric acid, water, and brine, then dried(MgSO₄), filtered and concentrated in vacuo to provide the amide as awhite solid (56 mg; 88%).

[0591]¹H NMR (CDCl₃, 400 MHz): δ11.24 (s, 1H), 8.33 (s, 1H), 7.88 (d,1H), 6.50 (d, 1H), 3.86 (br s, 4H), 3.32 (br s, 4H), 2.22 (s, 3H). LRMS(Electrospray, positive): Da/e 279.3 (m+1).

EXAMPLE 127 2-Acetylamino-4-morpholin-4-yl-benzoic acid

[0592] Prepared via the lithium hydroxide hydrolysis procedure ofExample 119 (with heat).

[0593]¹H NMR (d6-DMSO, 400 MHz): δ12.93 (s, 1H), 11.35 (s, 1H), 8.18 (s,1H), 7.81 (d, 1H), 6.68 (d, 1H), 3.73 (br s, 4H), 3.24 (br s, 4H), 2.12(s, 3H). LRMS (Electrospray, negative): Da/e 263.3 (m−1).

EXAMPLE 128 2-Methanesulfonylamino-4-morpholin-4-yl-benzoic acid methylester

[0594] To stirred solution of methyl 2-amino-4-morpholin-4-yl-benzoate(47 mg; 0.2 mmol) in CH₂Cl₂ (2 mL) was added Hunig's base (0.10 mL; 0.57mmol) and methanesulfonyl chloride (0.040 mL; 0.52 mmol) at roomtemperature under a nitrogen atmosphere. After stirring for 4 hours, thereaction was quenched with MeOH (0.5 mL) and diluted with EtOAc (25 mL).The resulting solution was washed with 0.5 N aqueous hydrochloric acid,water and brine, then dried (MgSO₄), filtered, and concentrated invacuo. The residue was purified via radial chromatography (2:1hexanes/EtOAc) to provide the ester-sulfonamide as a yellow solid (45mg; 72%).

[0595]¹H NMR (CDCl₃, 400 MHz): δ10.55 (s, 1H), 7.89 (d, 1H), 7.18 (d,1H), 6.55 (dd, 1H), 3.87 (s, 3H), 3.83 (t, 4H), 3.32 (t, 4H), 3.02 (s,3H). ¹³C NMR (CDCl₃, 100 MHz): δ168.5, 155.3, 142.8, 133.0, 108.5,105.9, 102.5, 66.7, 52.3, 47.3, 39.8. LRMS (Electrospray, negative):Da/e 313.1 (m−1).

EXAMPLE 129(2-N-Methyl-N-(2,2,2-trifluoroacetyl)amino]-4-morpholin-4-yl-benzoicacid methyl ester

[0596] To a stirred solution of(2-N-methyl-N-(2,2,2-trifluoroacetyl)amino)-4-morpholin-4-yl-2-benzoicacid (330 mg; 0.99 mmol) in dry DMF (5 mL) was added K₂CO₃ (140 mg; 1.01mmol) and iodomethane (0.065 mL; 1.04 mmol) at room temperature under anitrogen atmosphere. After stirring for 8 hours, the reaction wasdiluted with EtOAc (35 mL), washed with water (3×10 mL) and brine, thendried (MgSO₄), filtered, and concentrated in vacuo. The residue waspurified via radial chromatography (2 mm chromatotron plate with 2:1hexanes/EtOAc) to provide the N-methyl amide as a white solid (303 mg;88%).

[0597]¹H NMR (CDCl₃, 400 MHz): δ8.00 (d, 1H), 6.88 (dd, 1H), 6.67 (d,1H), 3.86 (t, 4H), 3.84 (d, 3H), 3.30 (t, 4H), 3.29 (s, 3H). LRMS(Electrospray, positive): Da/e 347.2 (m+1).

EXAMPLE 130 2-Methylamino-4-morpholin-4-yl-benzoic acid methyl ester

[0598] Prepared via the lithium hydroxide hydrolysis procedure ofExample 119 (without heat).

[0599]¹H NMR (CDCl₃, 400 MHz): δ7.78 (d, 1H), 6.16 (dd, 1H), 5.95 (d,1H), 3.84 (t, 4H), 3.80 (s, 3H), 3.28 (t, 4H), 2.89 (d, 3H). ¹³C NMR(CDCl₃, 100 MHz): δ168.9, 155.9, 153.6, 133.1, 102.4, 94.4, 67.0, 51.3,48.0, 29.8. LRMS (Electrospray, positive): Da/e 251.4 (m+1).

EXAMPLE 131 2-Methylamino-4-morpholin-4-yl-benzoic acid

[0600] Prepared via the lithium hydroxide procedure of Example 119 (withheat).

[0601]¹H NMR (d6-DMSO, 400 MHz): δ7.59 (d, 1H), 6.17 (dd, 1H), 5.94 (d,1H), 3.69 (t, 41H), 3.31 (br s, 1H), 3.21 (t, 4H), 2.80 (s, 3H). LRMS(Electrospray, negative): Da/e 235.0 (m−1).

EXAMPLE 132 2-Chloro-1-(2-acetamido-4-morpholin-4-yl-phenyl)-ethanone

[0602] A 25-mL round-bottom flask equipped with stir bar and nitrogeninlet was charged with 271.6 mg (1.07 mmol) of 2-amino-4-morpholinylchloro-acetophenone and the resulting solution was chilled (5° C.). Tothis was added 1 mL (10 mmol) of acetic anhydride. After 18 h, 1 mL MeOHwas added, and the reaction stirred for an additional 1 h, thenconcentrated in vacuo to a brown solid which was purified by radialchromatography (2 mm chromatotron plate). Solvent: 100% CH₂Cl₂ to 2%MeOH/CH₂Cl₂. Recovered 170.1 mg (53.7%) of the desired amide as a tansolid.

[0603]¹H NMR (CDCl₃, 400 MHz, mixture of rotomers): δ11.84 (s, 1H), 8.39(t, 1H), 7.66 (dd, 1H), 6.50 (dt, 1H), 4.65 (d, 2H), 3.82 (dt, 4H), 3.40(dt, 4H), 2.22 (d, 3H).

EXAMPLE 133 1-Acetyl-6-morpholin-4-yl-1,2-dihydro-indol-3-one

[0604] To a cooled (0° C.), stirred suspension of sodium hydride (5.3 mgof 60% dispersion in oil; 0.16 mmol) in dry tetrahydrofuran (2 mL) wasadded a solution ofN-(2-(2-chloroacetyl)-5-morpholin-4-ylphenyl]acetamide (30.8 mg; 0.10mmol) in dry tetrahydrofuran (2 mL) via syringe under nitrogenatmosphere. The resulting mixture was stirred at 0° C. for 1 hour, thenwarmed to room temperature and stirred for an additional 2 hours. Thereaction mixture was concentrated in vacuo, and the residue purified viaradial chromatography (1 mm chromatotron plate with 30% EtOAc inhexanes) to provide the oxoindole as a pinkish tan solid (12.0 mg; 46%).

[0605]¹H NMR (CDCl₃, 400 MHz): δ7.99 (d, 1H), 7.59 (d, 1H), 6.67 (dd,1H), 4.23 (s, 2H), 3.83 (t, 4H), 3.40 (t, 4H), 2.28 (s, 3H). ¹³C NMR(CDCl₃, 100 MHz): δ191.9, 168.7, 157.7, 156.1, 125.3, 110.8, 101.4,66.7, 57.1, 47.7, 24.8.

EXAMPLE 134 4-Morpholin-4-yl-2-nitrobenzoic acid methyl ester

[0606] Prepared via the nucleophilic addition of morpholine procedure ofExample 118.

[0607]¹H NMR (CDCl₃, 400 MHz): δ7.75 (d, 1H), 7.03 (d, 1H), 6.95 (dd,1H), 3.84 (t, 4H), 3.83 (s, 3H), 3.30 (t, 4H). ¹³C NMR (CDCl₃, 100 MHz):δ164.9, 153.6, 151.8, 132.3, 115.7, 114.1, 108.4, 66.5, 52.9, 47.5. LRMS(Electrospray, positive): Da/e 267.1 (m+1).

EXAMPLE 135 4-Morpholin-4-yl-2-nitrobenzoic acid

[0608] Prepared via the lithium hydroxide hydrolysis procedure ofExample 119 (with heat).

[0609]¹H NMR (d6-Acetone, 400 MHz): δ7.87 (d, 1H), 7.21 (d, 1H), 7.18(dd, 1H), 3.81 (t, 4H), 3.43 (t, 4H). ¹³C NMR (d6-Acetone, 100 MHz):δ205.4, 164.2, 154.3, 132.6, 114.9, 112.2, 107.9, 66.3, 47.3. LRMS(Electrospray, negative): Da/e 251.0 (m−1).

EXAMPLE 136 (4-Morpholin-4-yl-2-nitro)-N-(methylcarboxymethyl)-benzamide

[0610] Prepared via the EDC coupling procedure of Example 75.

[0611]¹H NMR (CDCl₃, 400 MHz): δ7.45 (d, 1H), 7.39 (d, 1H), 7.03 (dd,1H), 6.36 (br s, 1H), 4.22 (d, 2H), 3.87 (t, 4H), 3.80 (s, 3H), 3.27 (t,4H). LRMS (Electrospray, positive): Da/e 324.2 (m+1).

EXAMPLE 137 Benzyl 2-((4-benzyl)carbonyl]-5-morpholin-4-yl-benzenephosphate

[0612] To a stirred, cooled (0° C.) suspension of2-hydroxy-4-morpholin-4-ylphenyl 4-methoxyphenyl ketone (89 mg, 0.28mmol) in acetonitrile (1.5 mL) and CH₂Cl₂ (1 mL) was added 1H-tetrazole(76 mg, 1.1 mmol), followed by dropwise addition of dibenzyldiisopropylphosphoramidite (250 μL, 0.74 mmol). The reaction mixture wasstirred under nitrogen at 0° C. for two hours, then warmed to roomtemperature and diluted with CH₂Cl₂. The mixture was washed with icecold saturated aqueous NaHCO₃, water, and brine, then dried (MgSO₄),filtered, and concentrated in vacuo. The residue was purified by radialchromatography (2:1 hexane/EtOAc, 1 mm silica gel plate) to give a lightbrown oil (155 mg, 99.4%).

[0613] The above-described light brown oil (155 mg, 0.28 mmol) wasdissolved in tetrahydrofuran (3 mL) and stirred under nitrogen at −78°C. A solution of 30% hydrogen peroxide in water (1.2 mL) was dropwiseadded. The reaction mixture was warmed to room temperature, andcontinued stirred for 45 minutes. It then was extracted with diethylether. The organic layer was further washed with saturated aqueoussodium thiosulfate three times, water, saturated aqueous NaHCO₃, andbrine, then dried (MgSO₄), filtered, and concentrated in vacuo.Purification of the residue by radial chromatography (2:1 to 1:2hexane/EtOAc, 1 mm silica gel plate) yielded a pale yellow oil (126 mg,79.1%)

[0614]¹H NMR (CDCl₃, 400 MHZ): δ7.78 (dd, 1H) 7.77 (d, 1H), 7.42 (dd,1H), 7.36 (s, 1H), 7.32-7.24 (m, 5H), 7.22-7.15 (m, 4H), 6.92-6.84 (m,3H), 6.70 (dd, 1H), 4.86-4.75 (m, 4H), 3.79 (t, 4H), 3.79 (s, 3H), 3.15(t, 4H). LRMS (Electrospray, positive): Da/e 574.5 (m+1).

EXAMPLE 138 4-Methylphenyl 4-morpholin-4-yl-2-(phosphonooxy)phenylmethanone disodium salt

[0615] To a solution of the above-described diester (126 mg, 0.22 mmol)in MeOH (3 mL) and EtOAc (1 mL) was added 10% palladium on carbon (10mg). The reaction flask was purged with hydrogen three times. The blacksuspension then was stirred at room temperature under hydrogen, whichwas supplied by a balloon. Half an hour later, mass spectroscopyindicated the formation of the mono-debenzylated intermediate (negativeelectrospray, Da/e 482.2 found). The hydrogenation was allowed toproceed for another 1.5 h for complete debenzylation. The reactionsystem then was purged with nitrogen. The mixture was filtered through aNylon-66 membrane to remove the palladium on carbon.

[0616] To the above-described filtrate was added a solution of NaHCO₃(37 mg, 0.44 mmol) in water (2 mL) to convert the acid to a bis-sodiumsalt. The mixture was stirred for ten minutes, then concentrated invacuo to remove the volatile solvents. The residue then was lyophilizedfrom a mixture of water and 1,4-dioxane to yield a yellow solid (98 mg,100%). HPLC analysis showed no impurity.

[0617]¹H NMR (CD₃OD, 400 MHz): δ7.76 (d, 1H), 7.76 (dd, 1H), 7.45 (d,1H), 7.19 (dd, 1H), 6.96 (d, 1H), 6.96 (dd, 1H), 6.54 (dd, 1H), 3.86 (s,3H), 3.81 (t, 4H), 3.33 (t, 4H). LRMS (Electrospray, negative):Da/e392.1 (m−1 for the acid form).

EXAMPLE 139 5-Hydroxy-7-morpholin-4-yl-2-phenyl-chromen-4-one

[0618] A solution of trifluoromethanesulfonic acid5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl ester (200 mg, 0.518 mmol),morpholine (0.05 mL, 0.622 mmol),tris(dibenzylideneacetone)dipalladium(0) (9.4 mg, 0.010 mmol),racemic-2,2′bis(diphenylphosphino)-1,1′-binapthyl (19 mg, 0.030 mmol),and cesium carbonate (236 mg, 0.725 mmol) in toluene (5 mL) was heatedto reflux for 24 h. The mixture was allowed to cool to room temperature,filtered through a ¾″ silica gel (60 Å) plug, then the plug was washedwith EtOAc (40 mL). The resulting solution was concentrated under vacuumand purified via Biotage chromatography with gradient elution from 15%EtOAc/hexanes to 50% EtOAc/hexanes to yield 37 mg (22%) of5-hydroxy-7-morpholin-4-yl-2-phenyl-chromen-4-one. See, A. Echavarren etal., J. Am. Chem. Soc., 109, pp. 5478-5486 (1987). Rf=0.12 (25%EtOAc/hexanes).

[0619]¹H NMR (CDCl₃, 400 MHz) δ12.56 (s, 1H), 7.85 (d, 2H), 7.54-7.48(c, 3H), 6.61 (s, 1H), 6.37 (d, 1H), 6.29 (d, 1H), 3.85 (m, 4H), 3.34(m, 4H). ¹³C (CDCl₃, 100MHz) d 182.0, 163.7, 161.9, 158.3, 156.3, 131.8,129.2, 126.4, 106.1, 106.0, 104.2, 97.2, 91.7, 66.7, 47.5. LRMS(Electrospray, positive): Da/e 324.6 (m+1).

EXAMPLE 140 5-Hydroxy-2-phenyl-7-piperidin-1-yl-chromen-4-one

[0620] A solution of trifluoromethanesulfonic acid5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl ester (130 mg, 0.337 mmol),piperidine (0.040 mL, 0.40 mmol),tris(dibenzylideneacetone)dipalladium(0) (15 mg, 0.017 mmol),biphenyl-2-yl-di-tert-butyl-phosphane (20 mg, 0.067 mmol), andtripotassium phosphate (K₃PO₄) (143 mg, 0.674 mmol) in THF (2 mL) washeated to reflux for 24 h, then cooled to room temperature for 24 h. Thereaction mixture was filtered through a ¾″ silica gel (60 Å) plug. Theplug was washed with EtOAc (75 mL), and the filtrate concentrated. Theconcentrate was purified via Biotage chromatography eluting with 5%EtOAc/hexanes to yield 18 mg (17%) of5-hydroxy-2-phenyl-7-piperidin-1-yl-chromen-4-one.

[0621]¹H NMR (CDCl₃, 400 MHz) δ12.6 (s, 1H), 7.87 (m, 2H), 7.52-7.49 (c,3H), 6.59 (m, 1H), 6.36 (m, 1H), 6.28 (m, 1H), 3.41 (br s, 4H), 1.68 (brs, 6H). LRMS (Electrospray, positive): Da/e 322.5 (m+1).

EXAMPLE 141 Trifluoromethanesulfonic acid3,5-dihydroxy-4-oxo-2-phenyl-4H-chromen-7-yl ester

[0622] To a solution of galangin (100 mg, 0.00037 mmol) andtriethylamine (0.103 mL, 0.740 mmol) in CH₂Cl₂ (6 mL) was addedN-phenyltrifluoro-methanesulfonimide (132 mg, 0.37 mmol). After 24 h thereaction was concentrated to yield a yellow semisolid and purified viaBiotage chromatography with gradient elution from 100% hexanes to 25%EtOAc/hexanes to yield 91 mg (61%) of trifluoromethanesulfonic acid3,5-dihydroxy-4-oxo-2-phenyl-4H-chromen-7-yl ester. Tf is CF₃SO₂—.

[0623]¹H NMR (CDCl₃, 400 MHz) δ12.64 (s, 1H), 10.19 (s, 1H), 8.24 (d,2H), 7.59-7.51 (c, 41H), 6.97 (d, 1H).

EXAMPLE 142 3,5-Dihydroxy-7-morpholin-4-yl-2-phenyl-chromen-4-one

[0624] A solution of trifluoromethanesulfonic acid3,5-dihydroxy-4-oxo-2-phenyl-4H-chromen-7-yl ester (90 mg, 0.224 mmol),morpholine (0.024 mL, 0.269 mmol),tris(dibenzylideneacetone)dipalladium(0) (10 mg, 0.011 mmol),biphenyl-2-yl-di-tert-butyl-phosphane (13 mg, 0.045 mmol), and K₃PO₄ (67mg, 0.314 mmol) in THF (1.2 mL) was capped and heated to 85° C. After 9h, the reaction mixture was allowed to cool to room temperature, thenfiltered through a ½″ silica gel (60 Å) plug. The plug was washed withEtOAc (40 mL) and the filtrate concentrated. The concentrate waspurified via Biotage chromatography eluting with 100% CH₂Cl₂ to yield 7mg (9%) of 3,5-dihydroxy-7-morpholin-4-yl-2-phenyl-chromen-4-one.Rf=0.22 (100% CH₂Cl₂).

[0625]¹H NMR (CDCl₃, 400 MHz) δ11.52 (s, 1H), 8.18 (d, 2H), 7.54-7.45(c, 3H), 6.65(s, 1H), 6.38 (s, 1H), 6.32 (s, 1H), 3.84 (m, 4H), 3.38(m,4H). LRMS (APCI, positive): Da/e 340.4 (m+1).

EXAMPLE 143 Trifluoromethanesulfonic acid 4-acetyl-3,5-dihydroxy-phenylester

[0626] A suspension of 1-(2,4,6-trihydroxyphenyl)-ethanone hydrate (5.0g, 26.9 mmol), triethylamine (7.50 mL, 53.7 mmol) and crushed 4 Åmolecular sieves was stirred in CH₂Cl₂ (250 mL). After 3 h,N-phenyltrifluoromethane-sulfonimide (9.61 g, 26.9 mmol) was added in 10portions (about 1 g each) over 5 hours. After 24 h, the reaction wasfiltered through a silica gel ¾″ (60 Å) plug, and the plug washed with5% MeOH/CH₂Cl₂ (200 mL). The filtrate was concentrated under reducedpressure, and the resulting oil purified via Biotage chromatographyeluting with 15% EtOAc/hexanes to yield 590 mg (73%) oftrifluoro-methanesulfonic acid 4-acetyl-3,5-dihydroxy-phenyl ester.Rf=0.50 (30% EtOAc/hexanes).

[0627]¹H NMR (CDCl₃, 400 MHz) δ10.60 (s, 2H), 6.34 (s, 2H), 2.76 (s,3H). ¹³C NMR (CDCl₃, 100 MHz) δ205.5, 163.2, 153.9, 110.1, 101.8, 101.6,33.5.

EXAMPLE 144 1-(2,6-Dihydroxy-4-morpholin-4-yl-phenyl)-ethanone

[0628] A solution of trifluoromethanesulfonic acid4-acetyl-3,5-dihydroxy-phenyl ester (1.0 g, 3.33 mmol), morpholine (0.35mL, 3.99 mmol), tris-(dibenzylideneacetone)dipalladium(0) (153 mg, 0.167mmol), biphenyl-2-yl-di-tert-butyl-phosphane (198 mg, 0.666 mmol), andK₃PO₄ (1.41 mg, 6.66 mmol) in THF (2 mL) was heated to 85° C. in asealed tube for 2 h. The reaction mixture was allowed to cool to roomtemperature, and filtered through a ¾″ silica gel (60 Å) plug. The plugwas washed with EtOAc (75 mL) and the filtrate concentrated. Theconcentrate was purified via Biotage chromatography with gradientelution from 100% hexanes to 40% EtOAc/hexanes to yield 475 mg (60%) of1-(2,6-dihydroxy-4-morpholin-4-yl-phenyl)-ethanone. Rf=0.16 (50%EtOAc/hexanes).

[0629]¹H NMR (CDCl₃, 400 MHz) δ7.26 (s, 2H), 5.82 (s, 2H), 3.79 (c, 4H),3.26 (m, 4H), 2.65 (s, 3H). LRMS (Electrospray, positive): Da/e 238.3(m+1).

EXAMPLE 1454-(5-Hydroxy-7-morpholin-4-yl-4-oxo-4H-chromen-2-yl)-benzonitrile

[0630] To a suspension of1-(2,6-dihydroxy-4-morpholin-4-yl-phenyl)-ethanone (45 mg, 0.190 mmol)and K₂CO₃ (130 mg, 0.948 mmol) in acetone (3 mL) was added4-cyanobenzoyl chloride 33 mg, 0.20 mmol). The reaction vessel wassealed and heated to 60° C. for 14 h. After cooling to room temperature,water (about 2 mL) was added, and the mixture was allowed to stir forabout 10 min. The contents were transferred to a separatory funnel andextracted with EtOAc (3×15 mL). The organic layer was dried (MgSO₄) andconcentrated. The concentrate was suspended in 50% EtOAc/hexanes andfiltered to yield 1 mg (1%) of4-(5-hydroxy-7-morpholin-4-yl-4-oxo-4H-chromen-2-yl)-benzonitrile.

[0631]¹H NMR (CDCl₃, 400 MHz) δ12.40 (s, 1H), 7.98 (d, 1H), 7.81 (d,1H), 6.67 (s, 1H) 6.4 (s, 1H), 6.31 (s, 1H), 3.90 m, 4H), 3.37 (m, 4H).

EXAMPLE 1463-(5-Hydroxy-7-morpholin-4-yl-4-oxo-4H-chromen-2-yl)-benzonitrile

[0632] To a suspension of1-(2,6-dihydroxy-4-morpholin-4-yl-phenyl)-ethanone (61 mg, 0.257 mmol)and K₂CO₃ (178 mg, 1.29 mmol) in acetone (3 mL) was added 3-cyanobenzoylchloride (43 mg, 0.257 mmol). The reaction vessel was sealed and heatedto 60° C. for 2 d. After cooling to room temperature, water (about 2 mL)was added, and the mixture was allowed to stir for about 10 min. Thecontents were transferred to a separatory funnel containing water (5 mL)and extracted with EtOAc (3×15 mL). The organic layer was dried (MgSO₄)and concentrated. The concentrate was purified via Biotagechromatography with gradient elution from 100% hexanes to 30%EtOAc/hexanes to yield 6 mg (7%) of3-(5-hydroxy-7-morpholin-4-yl-4-oxo-4H-chromen-2-yl)-benzonitrile.Rf=0.33 (50% EtOAc/hexanes).

[0633]¹H NMR (CDCl₃, 400 MHz) δ12.40 (s, 1H), 8.20 (s, 1H), 8.05 (d,1H), 7.81 (d, 1H), 7.66 (dd, 1H), 6.64 (s, 1H), 6.41 (s, 1H), 6.31 (s,1H), 3.87 (m, 4H), 3.38 (m, 4H). LRMS (APCI, positive): Da/e 349.4(m+1).

EXAMPLE 147 5-Hydroxy-2-(4-methoxyphenyl)-7-morpholin-4-yl-chromen-4-one

[0634] To a suspension of1-(2,6-dihydroxy-4-morpholin-4-yl-phenyl)-ethanone (112 mg, 0.472 mmol)and K₂CO₃ (326 mg, 2.36 mmol) in acetone (3 mL) was added p-anisoylchloride (0.066 mL, 0.472 mmol). The reaction vessel was sealed andheated to 80° C. for 4 days. After cooling to room temperature, water(about 2 mL) was added, and the mixture was allowed to stir for about 15min. The contents were transferred to a separatory funnel containingwater (5 mL) and extracted with EtOAc (3×15 mL). The organic layer wasdried (MgSO₄) and concentrated. The concentrate was purified via Biotagechromatography with gradient elution from 100% hexanes to 30%EtOAc/hexanes to yield 9 mg (5%) of5-hydroxy-2-(4-methoxy-phenyl)-7-morpholin-4-yl-chromen-4-one. Me isCH₃—.

[0635]¹H NMR (CDCl₃, 400 MHz) δ12.67 (s, 1H), 7.83 (d, 2H), 7.00 (d,2H), 7.81 (d, 1H), 6.54 (s, 1H), 6.37(d, 1H), 6.29 (d, 1H), 3.89 (s,3H), 3.86 (m, 4H), 3.34 (m, 4H). LRMS (Electrospray, positive): Da/e354.6 (m+1).

EXAMPLE 148 5-Hydroxy-7-morpholin-4-yl-2-pyridin-3-yl-chromen-4-one

[0636] To a suspension of1-(2,6-dihydroxy-4-morpholin-4-yl-phenyl)-ethanone (86 mg, 0.362 mmol)and K₂CO₃ (250 mg, 1.81 mmol) in acetone (3 mL) was added nicotinoylchloride hydrochloride (0.71 mL, 0.399). The reaction vessel was sealedand heated to 85° C. for 24 h. After cooling to room temperature, water(about 2 mL) was added, and the mixture was allowed to stir for about 10min. The contents were transferred to a separatory funnel containingwater (5 mL) and extracted with EtOAc (3×15 mL). The organic layer wasdried (MgSO₄) and concentrated. The concentrate was purified via Biotagechromatography with gradient elution from 100% hexanes to 100% EtOAc toyield 14 mg (12%) of5-hydroxy-7-morpholin-4-yl-2-pyridin-3-yl-chromen-4-one. Rf=0.22 (100%EtOAc).

[0637]¹H NMR (CDCl₃, 400 MHz) δ12.44 (s, 1H), 9.12 (s, 1H), 8.76 (d,1H), 8.13 (d, 1H), 7.47 (m, 1H), 6.64 (s, 1H), 6.39 (d, 1H), 6.30 (s,1H), 3.86 (m, 4H), 3.36 (m, 4H). LRMS (Electrospray, positive): Da/e325.6 (m+1).

EXAMPLE 149 2-Hydroxy-1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone

[0638] 1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-ethanone was dissolved intriethylamine (12 mL), and trimethylsilyl chloride (1.60 mL, 12.6 mmol)was added dropwise while maintaining the temperature of the solutionbelow 35° C. A solution of sodium iodide (0.54 g, 3.62 mmol) dissolvedin acetonitrile (30 mL) was added dropwise without allowing thetemperature to rise above 35° C. The reaction was stirred at 22° C. for16 hours, then poured into ice water/hexanes. The layers were separatedand the aqueous layer was washed with hexanes (2×). The combinedorganics were dried over K₂CO₃ and concentrated in vacuo. This material,4-(3-trimethylsilanyloxy-4-(1-trimethylsilanyl-oxy-vinyl)-phenyl]-morpholine,(¹H NMR (CDCl₃, 400 MHz): δ7.42 (d, 1H), 6.53-6.49 (m, 1H), 6.33-6.31(d, 1H), 5.06 (s, 1H), 4.53 (s, 1H), 3.87-3.83 (m, 4H), 3.16-3.11 (m,4H), 0.28 (s, 9H), 0.23 (s, 9H)), was used in the reaction below.

[0639] 3-Chloroperoxybenzoic acid (1.48 g, 6.0 mmol) was slurried inhexanes (40 mL) and cooled to −78° C. A solution of4-(3-trimethylsilanyloxy-4-(1-trimethylsilanyloxy-vinyl)-phenyl]-morpholine(1.10 g, 3.01 mmol) dissolved in hexanes (5 mL) was added slowly. Theresulting suspension was maintained at −78° C. for 60 minutes thenslowly warmed to 22° C. After stirring at 22° C. for 16 hours, thereaction mixture was diluted with methanol and concentrated in vacuo.The residue was redissolved in methanol and concentrated two additionaltimes. The solids were resuspended in EtOAc and washed 2 times withsaturated NaHCO₃, and once with saturated NaCl then dried over Na₂SO₄.After concentration, the residue was chromatographed on SiO2 using 2:1hexane/EtOAc then 1:1 hexane/EtOAc. After concentration, the alcohol wasrecrystallized from EtOAc. (19% yield).

[0640]¹H NMR (CDCl₃, 400 MHz): δ11.86 (s, 1H), 7.38 (m, 1H), 6.40 (m,1H), 6.31 (s, 1H), 4.76 (d, 1H), 3.86-3.80 (m, 4H), 3.38-3.32 (m, 4H).¹³C NMR (CDCl₃, 100 MHz): d 199.1, 164.5, 157.0, 130.0, 108.7, 106.1,100.3, 66.5, 63.7, 47.0. LRMS (Electrospray, negative): Da/e 236.4(m−1).

[0641] Obviously, many modifications and variations of the invention ashereinbefore set forth can be made without departing from the spirit andscope thereof, and, therefore, only such limitations should be imposedas are indicated by the appended claims.

What is claimed is:
 1. A DNA-PK inhibitor having a formula

or a pharmaceutically acceptable salt thereof, wherein: n is an integer0 through 4; Z, independently, is CR³ or N; A is an optionallysubstituted four- to seven-membered aliphatic ring containing 0, 1, 2,or 3 heteroatoms, independently selected from the group consisting of N,O, and S; R¹ is selected from the group consisting of hydrogen, alkyl,substituted alkyl, cycloalkyl, heterocycloalkyl, N(R^(h))₂, OR^(h),carboxyl, carboxy, nitro, hydrazono, hydroxyamino, cyano, aldehyde,carboxamide, thiocarboxamide, acyl, mercapto, sulfonyl, trifluoromethyl,heteroaryl, and substituted heteroaryl; R² is selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, carbamoyl,carboxamide, N(R^(h))₂, carboxy, OR^(h), sulfamyl, nitro, phosphate, andsulfonamido; or R¹ and R² are taken together with the carbon atoms towhich each is attached to form a 5-, 6-, or 7-membered ring, wherein 1,2, or 3 carbon atoms of R¹ and R² optionally are a heteroatom selectedfrom the group consisting of O, N, S, and P, said ring optionallysubstituted with one or more ═O, ═S, ═NH, OR^(b), N(R^(h))₂, carboxyl,carboxy, alkyl, aryl, substituted aryl, heteroaryl, or substitutedheteroaryl, said heteroatom optionally substituted with a group selectedfrom the group consisting of aryl, substituted aryl, alkyl, alkylsubstituted with acyl, and acyl; R³, independently, is selected from thegroup consisting of hydrogen, halo, aldeyhde, OR^(h), nitro, N(R^(h))₂,carboxyl, carboxy, sulfonamido, sufamyl, and sulfo or a halidederivative thereof, wherein R^(h), independently, is selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl,aryl, substituted aryl, heteroaryl, and substituted heteroaryl; and R⁴,independently, is selected from the group consisting of OR^(h), halo,N(R^(h))₂, aldehyde, alkyl, substituted alkyl, acyl, aryl, substitutedaryl, heteroaryl, and substituted heteroaryl; with the proviso that whenA is morpholinyl, R² and R⁴ are hydrogen, and ZR³ is CH at eachoccurrence, then R¹ is different from —(CO)—CH₃, (C═CH₂)-phenyl, andnitro; and with the proviso that when A is morpholinyl, R⁴ is hydrogen,and Z is nitrogen at each occurrence, then R¹ and R², when takentogether, is different from triazole.
 2. The inhibitor of claim 1wherein A is selected from the group consisting of morpholinyl,piperazinyl, thiomorpholinyl, piperidinyl, and tetrahydropyranyl.
 3. Theinhibitor of claim 1 wherein R¹ is selected from the group consisting of—H, —NH₂, —(CO)—NH₂, —(CO)—NH—OH, —(CO)—NH—NH₂, —(CO)—NH—NH—R^(f),—(CO)—OH, —(CO)—O—CH₃, —(CO)—O—CH₂—CH₃, —(CO)—(4-methoxy)phenyl,—(CO)-(4-hydroxy)phenyl, —(CO)-(3-chlorophenyl), —(CO)-phenyl,—(CO)-benzyl, —(CO)—C₁₋₄alkyleneOR^(h), —(CO)—C₁₋₄alkyleneSR^(h),

—NO₂, —OH, —(CO)—C₁₋₄ alkyl, -cycloalkyl, —(CO)-substituted alkyl,—(CO)-(methoxy)alkyl, —(CO)-(alkoxy) substituted alkyl, —(CO)-aryl,—(CO)-heteroaryl, —(CO)-(substituted alkyl)_(p)-aryl, —(CO)-(substitutedalkoxy)_(p)-aryl, —(CO)—((NR^(k))_(p)-substituted alkoxy)-aryl,—(CO)aryl-R^(d), —(CO)-aryl-R^(e), —(CO)-aryl-R^(f), —CH═N—OH,—CH═N—NH₂, —CH═N—NH—CH₃, —CH═N—NH—CH₂-phenyl, —CF₃, —(CO)—CF₃,—(CO)—CH₂-morpholinyl, —(CO)—CH₂-heteroaryl, —(CO)—CH₂—CH—(CH₃)₂,—(CO)—CH₂—CH₂—(SO₂)—CH₃, —CHO, —C≡N, —CH₂—OH, —(CO)NR^(d)R^(e),—(CS)—NH₂, —(CO)—R^(f), —(CO)—CH₂Cl, —(CO)CH₂NR^(d)R^(e),—(CO)—CH₂—S—(CO)—CH₃, —(CO)—CH₂—SH, —(SO₂)-phenyl,2-(anilino)-4-thiazolyl-, 2-(pyridyl)-4-thiazolyl-, -benzoxazolyl,-imidazolyl, thiazolyl, -substituted thiazolyl, -benzimidazolyl,-benzothiazolyl, -tetrazolyl, -(N-benzyi)-tetrazolyl,-(N-methyl)-tetrazolyl, -pyrazolyl, -(N-benzyl)-pyrazolyl,-(N-methyl)-pyrazolyl, -(N-acetyl)-pyrazolyl, -(N-mesyl)-pyrazolyl,-pyrazolyl-(CO)—R^(u)R^(v), —(N-phenyl)-piperazinyl, -isoxazolyl,-pyrimidinyl, -(2-NH—CH₂-phenyl)-pyrimidinyl,-(2-(SO)-methyl)-pyrimidinyl,-(2-N—(N-t-butoxycarbonyl)-piperazinyl)-pyrimidinyl, and-(2-NH—CH₂-pyridine)-pyrimidinyl; wherein R^(d) is selected from thegroup consisting of —H, -alkyl, —CH₂-phenyl, -phenyl, —O—CH₃, -pyridyl,-thiazolyl, -thiazinyl, —O—CH₂-phenyl, —O-phenyl, —O-methoxyphenyl, —OH,—CH₂—(CO)—O—CH₃, and —CH₂—(CO)—OH; R^(e) is selected from the groupconsisting of —H, —CH₂—CH₂—O—CH₃, —CH₂—CH₂—CH₂—N(CH₃)₂, —CH₃,—CH₂—CH₂—(SO₂)—CH₃, —OCH₃, —CH₂-pyridyl, —CH₂-phenyl, -alkyl,—CH₂—(CO)—O—CH₃, and -cylcopropyl; or R^(d) and R^(e) are taken togetherto form -morpholinyl, -phenylpiperazinyl, -imidazolyl, -pyrrolidinyl,—(N-methyl)-piperazinyl, and -piperidinyl; R^(f) is selected from thegroup consisting of -phenyl, -phenyl-(CF₃), -methylphenyl,-methoxyphenyl, -pyridyl, -alkyl, -benzyl, -thiophenyl, -thiazolyl,-chlorophenyl, —C(═NH)—NH₂, -fluorophenyl, —(CO)-phenyl, —(CH₂)-phenyl;R^(u) is selected from the group consisting of —H, and -alkyl; R^(v) isselected from the group consisting of —O—(CO)—CH₃, —NH-t-butoxycarbonyl,—O-phenyl, and —O—CH₂-phenyl; or R^(u) and R^(v) are taken together withthe carbon atoms to which they are attached to form a 5-membered ringcontaining an N, said N optionally protected with t-butoxycarbonyl; R²is selected from the group consisting of —H, —OH, -Halo, —CH₂—OH,—(CO)—NH₂, —NH₂, —(CO)—O—CH₃, —O—CH₃, —NH—(CO)—CF₃, —NH—(CO)—CH₃,—NH—(SO₂)—CH₃, —NH—CH₃, —N(CH₃)—(CO)—CF₃, —N═((CH(phenyl)-CH₂—(CO)OH,—NO₂, —O—PO₃ ^(═), —O-alkyl, —O—(CH₂)_(p)—OH, —O—(CH₂)_(p)—O-benzyl,—O—(CO)-heteroaryl, —O—(CO)-amino acid, —O—(CO)-nicotinic acid,—O—(CO)-aryl, —O—(CO)-alkyl, —O—CH₂—(CO)-benzyl, —O—(SO₂)—O—CF₃,—(CH₂)—CH═CH═N(CH₃)₂, —O—(SO₃)—, and —O—(PO)(OR^(j))(OR^(k); whereinR^(j) independently are H, aryl, alkyl, or heterocyclic; or R¹ and R²are taken together to form a three- or four-membered component,respectively, of a five- or six-membered ring, preferably said ringselected from the group consisting of -2-imidazolidonyl-,—R^(g)-thiazolyl-, -carbonylpyrrolyl methyl ketone-,-4-imino-1,3,2,-oxathiaphosphanyl-2-thione-,-4-imino-1,3,2,-oxathiaphosphanyl-2-thione-2-(4′-methoxy)phenyl-,-3-oxofuranyl-, —N-acetyl-3-oxopyrrolinyl-, —N—(CH₂—COOH)-quinolonyl-,—N-(t-butoxycarbonyl)-quinolonyl-, —N—(CH₂—COOH)-quinolinyl-,—N-(t-butoxycarbonyl)-quinolinyl-, and

wherein B is aryl or a nitrogen-containing heteroaryl, R⁹ is H orOR^(h), and R¹⁰ is selected from the group consisting of halo, OR^(h),O(CH₂)₁₋₃N(R^(h))₂, O(CH₂)₁₋₃CO₂H, CN, morphohinyl, andN-(4-methyl)-piperazinyl; wherein R^(g) is selected from the groupconsisting of -pyridyl and -anilino; R³, independently, is selected fromthe group consisting of —H, —OH, —OR^(d), —NO₂, —NH₂, NH—R^(d), -halo,—CHO, —(SO₂)—OH, —(SO₂)—Cl and —(SO₂)—NR^(i)R^(k); wherein R^(i) isselected from the group consisting of —H, —CH₃, —CH₂-phenyl, -phenyl,—CH₂—CH₂—O—CH₃, —CH₂—CH₂—CH₂—N(CH₃)₂, —O—CH₃, —CH₂—CH₂—(SO₂)—CH₃,-pyridyl, -thiazolyl, —O—CH₂-phenyl, —OH, —CH₂—(CO)—O—CH₃, and—CH₂—(CO)—OH; R^(k) is selected from the group consisting of —H, —O—CH₃,—CH₂-pyridyl, —CH₂-phenyl, —CH₃, —CH₂—(CO)—O—CH₃, -cyclopropyl, and—CH₂-cyclopropyl; or R^(i) and R^(k) are taken together to formmorpholinyl, phenylpiperazinyl, imidazolyl, pyrrolidinyl,(N-methyl)-piperazinyl, and piperidinyl; and R⁴, independently, isselected from the group consisting of —H, —CH₃, —OCH₃, —OH, —(CO)—CH₃,-methoxyphenyl, and -pyridinyl.
 4. The inhibitor of claim 1 wherein R¹is selected from the group consisting of —H, —OH, —NH₂, —CH₂OH, —C≡N,—(CO)—NH₂, —(CO)—OH, —(CO)—O—CH₃, —CH═N—OH, —CH═N—NH₂, —CH═N—NH—CH₃,—(CO)—CF₃,—(CO)H, —NO₂, —(CO)-alkyl, —(CO)-substituted alkyl,—(CO)-aryl, —(CO)-substituted aryl, —(CO)-heteroaryl,—(CO)—CH₂—NR^(d)R^(e), and —(CO)NR^(d)R^(e).
 5. The inhibitor of claim 1wherein R² is —H, —OH, —F, —CH₂—OH, —NH₂, —NH—(CO)—CF₃, —NH—(CO)—CH₃,—NH—(SO₂)—CH₃, —NH—CH₃, and —N(CH₃)—(CO)—CF₃.
 6. A DNA-PK inhibitorhaving a formula:

or a pharmaceutically acceptable salt thereof, wherein: Z,independently, is CR⁷ or N; L is selected from the group consisting ofalkylene, substituted alkylene, carbonyl, carbamoyl, NR^(h), oxy (—O—),thio (—S—), thionyl (—SO—), and sulfonyl; A is absent, or A is anoptionally substituted four- to seven-membered aliphatic ring containing0, 1, 2, or 3 heteroatoms, independently selected from the groupconsisting of N, O, and S; R⁵ is selected from the group consisting ofhydrogen, alkyl, substituted alkyl, cycloalkyl, heterocycloalkyl,N(R^(h))₂, OR^(h), carboxyl, carboxy, nitro, hydrazono, hydroxyamino,cyano, aldehyde, carboxamide, thiocarboxamide, acyl, mercapto, sulfonyl,trifluoromethyl, heteroaryl, and substituted heteroaryl; R⁶ is selectedfrom the group consisting of hydrogen, alkyl, substituted alkyl,carbamoyl, carboxamide, N(R^(h))₂, carboxy, OR^(h), sulfamyl, nitro,phosphate, and sulfonamido; or R⁵ and R⁶ are taken together with thecarbon atoms to which each is attached to form a 5-, 6-, or 7-memberedring, wherein 1, 2, or 3 carbon atoms of R⁵ and R⁶ optionally are aheteroatom selected from the group consisting of O, N, S, and P, saidring optionally substituted with one or more of ═O, ═S, ═NH, OR^(h),N(R^(h))₂, carboxyl, carboxy, alkyl, aryl, substituted aryl, heteroaryl,or substituted heteroaryl, and said heteroatom optionally substitutedwith a substituent selected from the group consisting of aryl,substituted aryl, alkyl, alkyl substituted with acyl, and acyl; R⁷,independently, is selected from the group consisting of hydrogen, halo,aldehyde, OR^(h), nitro, N(R^(h))₂, carboxyl, carboxy, sulfamyl,sulfonamido, and sulfo or a halide derivative thereof, wherein R^(h),independently, is selected from the group consisting of hydrogen, alkyl,substituted alkyl, cycloalkyl, aryl, substituted aryl, heteroaryl, andsubstituted heteroaryl; and R⁸, independently, is selected from thegroup consisting of OR^(h), halo, N(R^(h))₂, aldehyde, alkyl, subtitutedalkyl, acyl, aryl, substituted aryl, heteroaryl, and substitutedheteroaryl.
 7. The inhibitor of claim 6 wherein R⁵ is selected from thegroup consisting of —H, —NH₂, —(CO)—NH₂, —(CO)—NH—OH, —(CO)—NH—NH₂,—(CO)—NH—NH—R^(f), —(CO)—OH, —(CO)—O—CH₃, —(CO)—O—CH₂—CH₃,—(CO)-(4-methoxy)phenyl, —(CO)-(4-hydroxy)phenyl,—(CO)-(3-chlorophenyl), —(CO)-phenyl, —(CO)-benzyl,—(CO)—C₁₋₄alkyleneOR^(h), —(CO)—C₁₋₄alkyleneSR^(h),

—NO₂, —OH, —(CO)—C₁₋₄ alkyl, -cycloalkyl, —(CO)-substituted alkyl,—(CO)-(methoxy)alkyl, —(CO)-(alkoxy) substituted alkyl, —(CO)-aryl,—(CO)-heteroaryl, —(CO)-(substituted alkyl)_(p)-aryl, —(CO)-(substitutedalkoxy)_(p)-aryl, —(CO)—((N^(k))_(p)-substituted alkoxy)-aryl,—(CO)aryl-R^(d), —(CO)-aryl-R^(e), —(CO)-aryl-R^(f), —CH═N—OH,—CH═N—NH₂, —CH═N—NH—CH₃, —CH═N—NH—CH₂-phenyl, —CF₃, —(CO)—CF₃,—(CO)—CH₂-morpholinyl, —(CO)—CH₂-heteroaryl, —(CO)—CH₂—CH—(CH₃)₂,—(CO)—CH₂—CH₂—(SO₂)—CH₃, —CHO, —C≡N, —CH₂—OH, —(CO)NR^(d)R^(e),—(CS)—NH₂, —(CO)—R^(f), —(CO)—CH₂Cl, —(CO)—CH₂NR^(d)R^(e),—(CO)—CH₂—S—(CO)—CH₃, —(CO)—CH₂—SH, —(SO₂)-phenyl,2-(anilino)-4-thiazolyl-, 2-(pyridyl)-4-thiazolyl-, -benzoxazolyl,-imidazolyl, -thiazolyl, -substituted thiazolyl, -benzimidazolyl,-benzothiazolyl, -tetrazolyl, —(N-benzyl)-tetrazolyl,—(N-methyl)-tetrazolyl, -pyrazolyl, —(N-benzyl)-pyrazolyl,—(N-methyl)-pyrazolyl, —(N-acetyl)-pyrazolyl, —(N-mesyl)-pyrazolyl,-pyrazolyl-(CO)—R^(u)R^(v), —(N-phenyl)-piperazinyl, -isoxazolyl,-pyrimidinyl, -(2-NH—CH₂-phenyl)-pyrimidinyl,-(2-(SO)-methyl)-pyrimidinyl,-(2-N-(N-t-butoxycarbonyl)-piperazinyl)-pyrimidinyl, and-(2-NH—CH₂-pyridine)-pyrimidinyl; wherein R^(d) is selected from thegroup consisting of —H, -alkyl, —CH₂-phenyl, -phenyl, —O—CH₃, -pyridyl,-thiazolyl, -thiazinyl, —O—CH₂-phenyl, —O-phenyl, —O-methoxyphenyl, —OH,—CH₂—(CO)—O—CH₃, and —CH₂—(CO)—OH; R^(e) is selected from the groupconsisting of —H, —CH₂—CH₂—O—CH₃, —CH₂—CH₂—CH₂—N(CH₃)₂, —CH₃,—CH₂—CH₂—(SO₂)—CH₃, —O—CH₃, —CH₂-pyridyl, —CH₂-phenyl, -alkyl,—CH₂—(CO)—O—CH₃, and -cylcopropyl; or R^(d) and R^(e) are taken togetherto form -morpholinyl, -phenylpiperazinyl, -imidazolyl, -pyrrolidinyl,—(N-methyl)-piperazinyl, and -piperidinyl; R^(f) is selected from thegroup consisting of -phenyl, -phenyl-(CF₃), -methylphenyl,-methoxyphenyl, -pyridyl, -alkyl, -benzyl, -thiophenyl, -thiazolyl,-chlorophenyl, —C(═NH)—NH₂, -fluorophenyl, —(CO)-phenyl, —(CH₂)-phenyl;R^(u) is selected from the group consisting of —H, and -alkyl; R^(v) isselected from the group consisting of —O—(CO)—CH₃, —NH-t-butoxycarbonyl,—O-phenyl, and —O—CH₂-phenyl; or R^(u) and R^(v) are taken together withthe carbon atoms to which they are attached to form a 5-membered ringcontaining an N, said N optionally protected with t-butoxycarbonyl; R⁶is selected from the group consisting of —H, —OH, -Halo, —CH₂—OH,—(CO)—NH₂, —NH₂, —(CO)—O—CH₃, —O—CH₃, —NH—(CO)—CF₃, —NH—(CO)—CH₃,—NH—(SO₂)—CH₃, —NH—CH₃, —N(CH₃)—(CO)—CF₃, —N═((CH(phenyl)-CH₂—(CO)OH,—NO₂, —O—PO₃ ^(═), —O-alkyl, —O—(CH₂)_(p)—OH, —O—(CH₂)_(p)—O-benzyl,—O—(CO)-heteroaryl, —O—(CO)-amino acid, —O—(CO)-nicotinic acid,—O—(CO)-aryl, —O—(CO)-alkyl, —O—CH₂—(CO)-benzyl, —O—(SO₂)—O—CF₃,—(CH₂)—CH═CH═N(CH₃)₂, —O—(SO₃)—, and —O—(PO)(OR^(j))(OR^(k)); whereinR^(j) independently are H, aryl, alkyl, or heterocyclic; or R⁵ and R⁶are taken together to form a three- or four-membered component,respectively, of a five- or six-membered ring, preferably said ringselected from the group consisting of -2-imidazolidonyl-,—R^(g)-thiazolyl-, -carbonylpyrrolyl methyl ketone-,-4-imino-1,3,2,-oxathiaphosphanyl-2-thione-,-4-imino-1,3,2,-oxathiaphosphanyl-2-thione-2-(4′-methoxy)phenyl-,-3-oxofuranyl-, -N-acetyl-3-oxopyrrolinyl-, —N—(CH₂—COOH)-quinolonyl-,—N-(t-butoxycarbonyl)-quinolonyl-, —N—(CH₂—COOH)-quinolinyl-,—N-(t-butoxycarbonyl)-quinolinyl-, and

wherein B is aryl or a nitrogen-containing heteroaryl, R⁹ is H orOR^(h), and R¹⁰ is selected from the group consisting of halo, OR^(h),O(CH₂)₁₋₃N(R^(h))₂, O(CH₂)₁₋₃CO₂H, CN, morpholinyl, andN-(4-methyl)-piperazinyl; wherein R^(g) is selected from the groupconsisting of -pyridyl and -anilino; R⁷, independently, is selected fromthe group consisting of —H, —OH, OR^(d), —NO₂, —NH₂, —NH—R^(d), -halo,—CHO, —(SO₂)—OH, —(SO₂)—Cl, and —(SO₂)—NR^(i)R^(k); wherein R^(i) isselected from the group consisting of —H, —CH₃, —CH₂-phenyl, -phenyl,—CH₂—CH₂—O—CH₃, —CH₂—CH₂—CH₂—N(CH₃)₂, —O—CH₃, —CH₂—CH₂—(SO₂)—CH₃,-pyridyl, -thiazolyl, —O—CH₂-phenyl, —OH, —CH₂—(CO)—O—CH₃, and—CH₂—(CO)—OH; R^(k) is selected from the group consisting of —H, —O—CH₃,—CH₂-pyridyl, —CH₂-phenyl, —CH₃, —CH₂—(CO)—O—CH₃, -cyclopropyl, and—CH₂-cyclopropyl; or R^(i) and R^(k) are taken together to formmorpholinyl, phenylpiperazinyl, imidazolyl, pyrrolidinyl,(N-methyl)-piperazinyl, and piperidinyl; and R⁸, independently, isselected from the group consisting of —H, —CH₃, —OCH₃, —OH, —(CO)—CH₃,-methoxyphenyl, and -pyridinyl.
 8. The inhibitor of claim 6 wherein R⁵is selected from the group consisting of —H, —OH, —NH₂, —CH₂OH, —C≡N,—(CO)—NH₂, —(CO)—OH, —(CO)—O—CH₃, —CH═N—OH, —CH═N—NH₂, —CH═N—NH—CH₃,—(CO)—CF₃,—(CO)H, —NO₂, —(CO)-alkyl, —(CO)-substituted alkyl,—(CO)-aryl, —(CO)-substituted aryl, —(CO)-heteroaryl,—(CO)—CH₂—NR^(d)R^(e), and —(CO)NR^(d)R^(e).
 9. The inhibitor of claim 6wherein R⁶ is selected from the group consisting of —H, —OH, —F,—CH₂—OH, —NH₂, —NH—(CO)—CF₃, —NH—(CO)—CH₃, —NH—(SO₂)—CH₃, —NH—CH₃, and—N(CH₃)—(CO)—CF₃.
 10. A DNA-PK inhibitor selected from the groupconsisting of: benzyl 2-((4-benzyl)carbonyl)-5-morpholin-4-yl-benzenephosphate; 4-methylphenyl 4-morpholin-4-yl-2-(phosphonooxy)phenylmethanone disodium salt; 5-morpholin-4-yl-2-nitrophenylamine;5-(4-methyl-piperazin-1-yl)-2-nitrophenylamine;2-hydroxymethyl-5-morpholin-4-yl-phenol;2-nitro-5-thiomorpholin-4-yl-phenylamine;N¹-morpholin-4-yl-4-nitrobenzene-1,3-diamine;1-(3-amino-4-nitrophenyl)-piperidin-4-ol;2-nitro-5-piperidin-1-yl-phenylamine;5-(4-acetyl-piperazin-1-yl)-2-nitrophenylamine;2-nitro-5-piperazin-1-yl-phenylamine;1-(3-amino-4-nitrophenyl)-piperidin-3-ol;N¹-(2-morpholin-4-yl-ethyl)-4-nitrobenzene-1,3-diamine;5-(4-(2-methoxyphenyl)-piperazin-1-yl]-2-nitrophenylamine;5-(cis-2,6-dimethylmorpholin-4-yl)-2-nitrophenylamine;2-nitro-5-(4-pyridin-2-yl-piperazin-1-yl)-phenylamine;N¹-(3-morpholin-4-yl-propyl)-4-nitrobenzene-1,3-diamine;2-hydroxy-4-morpholin-4-yl-benzonitrile;(5-morpholin-4-yl-2-nitrophenyl)-methanol;2-hydroxy-4-morpholin-4-yl-benzoic acid;2-hydroxy-4-morpholin-4-yl-benzoic acid methyl ester;5-morpholin-4-yl-2-nitro-benzamide;2-hydroxy-4-morpholin-4-yl-benzaldehyde;5-morpholin-4-yl-2-nitro-phenol;1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone;1-(2-hydroxy-4-morpholin-4-yl-phenyl)-propan-1-one;1-(2-hydroxy-4-morpholin-4-yl-phenyl)-3-methyl-butan-1-one;1-(2-hydroxy-4-morpholin-4-yl-phenyl)-1-phenyl-methanone;2,2,2-trifluoro-1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone;4-amino-2-morpholin-4-yl-pyrimidine-5-carboxylic acid;1-(5-bromo-2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone;1-(3-bromo-2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone;1-(3,5-dichloro-2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone;1-(3-chloro-2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone;1-(5-fluoro-2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone;1-(3-fluoro-2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone;1-(2-hydroxy-4-(tetrahydropyran-4-yloxy)-phenyl]-ethanone;5-(morpholin-4-yl)-1,3-dihydro-benzimidazol-2-one;2-methoxy-4-morpholin-4-yl-benzaldehyde;4-methoxy-6-morpholin-4-yl-benzene-1,3-dicarbaldehyde;2-hydroxy-5-morpholin-4-yl-benzoic acid methyl ester;2-((hydroxyimino)methyl)-5-morpholin-4-yl-phenol;2-hydrazonomethyl-5-morpholin-4-yl-phenol;2-hydroxy-4-((1-morpholin-4-yl-methanoyl)-amino]-benzoic acid;2-hydroxy-4-morpholin-4-ylmethyl-benzoic acid methyl esterhydrochloride; 2-hydroxy-4-morpholin-4-ylmethyl-benzoic acidtrifluoroacetate; 2-hydroxy-4-morpholin-4-ylmethyl benzoic acidhydrochloride; 4-amino-2-hydroxy-benzoic acid methyl ester;2-hydroxy-4-morpholin-4-yl-benzoic acid methyl ester;2-hydroxy-N-methyl-4-morpholin-4-yl-benzamide;1-(2-hydroxy-4-morpholin-4-yl-phenyl)-1-morpholin-4-yl-methanone;2-hydroxy-4-morpholin-4-yl-benzamide;2-hydroxy-4-morpholin-4-yl-N-benzyl-benzamide;2-hydroxy-4-morpholin-4-yl-N-phenyl-benzamide;N-cyclopropyl-2-hydroxy-4-morpholin-4-yl-N-phenyl-benzamide;2-hydroxy-N-(2-methoxy-ethyl)-4-morpholin-4-yl-benzamide;2-hydroxy-4-morpholin-4-yl-N-methoxy-N-methyl-benzamide;2-hydroxy-4-morpholin-4-yl-N-(3-dimethylamino-propyl)-benzamide;2-hydroxy-N-methoxy-4-morpholin-4-yl-benzamide;2-hydroxy-N-(2-methanesulfonyl-ethyl)-4-morpholin-4-yl-benzamide;2-hydroxy-4-morpholin-4-yl-N-pyridin-3-yl-benzamide;2-hydroxy-4-morpholin-4-yl-N-pyridin-4-yl-benzamide;2-hydroxy-4-morpholin-4-yl-N-thiazol-2-yl-benzamide;2-hydroxy-4-morpholin-4-yl-N-( 1,4-thiazin-2-yl)-benzamide;2,N-dihydroxy-4-morpholin-4-yl-benzamide;2-hydroxy-4-morpholin-4-yl-N-(4-pyridylmethyl)-benzamide;1-(2-hydroxy-4-morpholin-4-yl-phenyl)-1-(4-phenylpiperizin-1-yl)-methanone;2-hydroxy-4-morpholin-4-yl-benzoic acid;N-carboxymethyl-2-hydroxy-4-morpholin-4-yl-phenyl)-carboxamide methylester; N-carboxymethyl-2-hydroxy-4-morpholin-4-yl-phenyl-carboxamide;2-hydroxy-4-morpholin-4-yl-thiobenzamide;2-(4-ethylphenyl)-4-imino-7-morpholin-4-yl-benzo(e)-1,3,2-oxathiaphos-phane-2-thione;1-(2-hydroxy-4-morpholin-4-yl-phenyl)-1-phenyl-methanone;1-(2-hydroxy-4-morpholin-4-yl-phenyl)-1-(4-trifluoromethylphenyl)-methanone;1-(2-hydroxy-4-morpholin-4-yl-phenyl)-1-(o-tolyl)-methanone;1-(2-hydroxy-4-morpholin-4-yl-phenyl)-1-(4-methoxyphenyl)-methanone;1-(2-hydroxy-4-morpholin-4-yl-phenyl)-1-pyridin-3-yl-methanone;1-(2-hydroxy-4-morpholin-4-yl-phenyl)-pentan-1-one;1-(2-hydroxy-4-morpholin-4-yl-phenyl)-2-phenyl-ethanone;1-(2-hydroxy-4-morpholin-4-yl-phenyl)-1-thiophen-2-yl-methanone;2-hydroxy-4-morpholin-4-yl-phenyl-1,3-thiazol-2-yl ketone;1-(3-chlorophenyl)-1-(2-hydroxy-4-morpholin-4-yl-phenyl)-methanone;2-chloro-1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone;1-(2-hydroxy-4-morpholin-4-yl-phenyl)-2-morpholin-4-yl-ethanone;1-(2-hydroxy-4-morpholin-4-yl-phenyl)-2-imidazol-1-yl-ethanone;1-(2-hydroxy-4-morpholin-4-yl-phenyl)-1-pyrrolidin-1-yl-methanone;1-(2-hydroxy-4-morpholin-4-yl-phenyl)-1-(4-methylpiperazin-1-yl)-methanone;2-hydroxy-4-morpholin-4-yl-phenyl-1-piperidin-1-yl-methanone;2-(benzyl-methyl-amino)-1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone;2-acetylthio-1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone;1-(2-hydroxy-4-morpholin-4-yl-phenyl)-2-mercapto-ethanone;6-morpholin-4-yl-2-hydrobenzo[b]furan-3-one;4-(2-methyl-4-morpholin-4-yl-phenyl)-2-(3-pyridyl)-1,3-thiazole;5-morpholin-4-yl-2-(2-phenylamino-1,3-thiazol-4-yl)-phenol;3-methoxy-1-morpholin-4-yl-benzene;4-methoxy-2-morpholin-4-yl-benzenesulfonic acid;4-methoxy-2-morpholin-4-yl-benzenesulfonyl chloride;4-methoxy-N-methyl-2-morpholin-4-yl-benzenesulfonamide;4-methoxy-2-morpholin-4-yl-N-benzyl-benzenesulfonachide;4-methoxy-2-morpholin-4-yl-N-cyclopropylmethyl-benzenesulfonamide; NNdiethyl(3 morpholin-4-yl-phenoxy)carboxamide;N,N-diethyl-(2-benzenesulfonyl-5-morpholin-4-yl-phenoxy)carboxamide;2-benzenesulfonyl-5-morpholin-4-yl-phenol;3-nitro-1-morpholin-4-yl-benzene; 3-morpholin-4-yl-phenylamine;1-(2-amino-4-morpholin-4-yl-phenyl)-2-chloro-ethanone;2-amino-4-morpholin-4-yl-N-benzyl-N-methyl-benzamide;1-(2-amino-4-morpholin-4-yl-phenyl)-1-pyrrolidin-1-yl-methanone;(2-amino-4-morpholin-4-yl-phenyl)-1-piperidin-1-yl-methanone;2-amino-4-fluorobenzoic acid methyl ester;4-fluoro-2-(2,2,2-trifluoroacetylamino)-benzoic acid methyl ester;4-morpholin-4-yl-2-(2,2,2-trifluoroacetylamino)-benzoic acid methylester; 2-amino-4-morpholin-4-yl-benzoic acid;2-methylsulfonylamino-4-morpholin-4-yl-benzoic acid;4-morpholin-4-yl-2-(2,2,2-trifluoroacetylamino)-N-benzyl-benzamide;N,N-dimethyl-4-morpholin-4-yl-2-(2,2,2-trifluoroacetylamino)-benzamide;2-amino-4-morpholin-4-yl-N,N-dimethyl-benzamide;N-methyl-4-morpholin-4-yl-2-(2,2,2-trifluoroacetylamino)-benzamide;2-amino-4-morpholin-4-yl-benzoic acid methyl ester;2-acetylamino-4-morpholin-4-yl-benzoic acid methyl ester;2-acetylamino-4-morpholin-4-yl-benzoic acid;2-methanesulfonylamino-4-morpholin-4-yl-benzoic acid methyl ester;(2-N-methyl-N-(2,2,2-trifluoroacetyl)amino)-4-morpholin-4-yl-benzoicacid methyl ester; 2-methylamino-4-morpholin-4-yl-benzoic acid methylester; 2-methylamino-4-morpholin-4-yl-benzoic acid;2-chloro-1-(2-acetamido-4-morpholin-4-yl-phenyl)-ethanone;1-acetyl-6-morpholin-4-yl-1,2-dihydro-indol-3-one;4-morpholin-4-yl-2-nitro-benzoic acid methyl ester;4-morpholin-4-yl-2-nitro-benzoic acid;4-morpholin-4-yl-2-nitrophenyl)N(methylcarboxymethyl)benzamide;5-hydroxy-7-morpholin-4-yl-2-phenyl-chromen-4-one;5-hydroxy-2-phenyl-7-piperidin-1-yl-chromen-4-one;trifluoromethanesulfonic acid3,5-dihydroxy-4-oxo-2-phenyl-4H-chromen-7-yl ester;3,5-dihydroxy-7-morpholin-4-yl-2-phenyl-chromen-4-one;trifluoromethanesulfonic acid 4-acetyl-3,5-dihydroxy-phenyl ester;1-(2,6-dihydroxy-4-morpholin-4-yl-phenyl)-ethanone;4-(5-hydroxy-7-morpholin-4-yl-4-oxo-4H-chromen-2-yl)-benzonitrile;3-(5-hydroxy-7-morpholin-4-yl-4-oxo-4H-chromen-2-yl)-benzonitrile;5-hydroxy-2-(4-methoxyphenyl)-7-morpholin-4-yl-chromen-4-one;5-hydroxy-7-morpholin-4-yl-2-pyridin-3-yl-chromen-4-one;2-hydroxy-1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone; and2-hydroxy-1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone.
 11. Apharmaceutical composition comprising (a) DNA-PK inhibitor claim 1 orclaim 6, and (b) a pharmaceutically acceptable carrier or diluent.
 12. Apharmaceutical composition comprising (a) a DNA-PK inhibitor of claim 1or 6, and (b) an anti-neoplastic agent.
 13. The pharmaceuticalcomposition of claim 12, with the proviso that when A is morpholinyl, Lis absent, R² and R⁴ are hydrogen, and ZR³ is CH at each occurrence,then R¹ is different from —(CO)—CH₃, —(C═CH₂)-phenyl, and nitro; andwith the proviso that when A is morpholinyl, R⁴ is hydrogen, and Z isnitrogen at each occurrence, then R¹ and R², when taken together, aredifferent from triazole.
 14. The pharmaceutical composition of claim 12wherein A is a morpholinyl, and L is absent.
 15. The pharmaceuticalcomposition of claim 12 wherein n is an integer from 0 through 4; Z,independently, is CR³ or N, or CR⁷ or N; L is absent, or L is selectedfrom the group consisting of —(CH₂)_(p)—, —(CHR^(k))_(p)—,—NR^(k)—(CHR^(k))_(p)—, —(CHR^(k))—NR^(k)—, —NR^(k)—, —C(═O)—, —O—,—NR^(k)—(CO)—, —(CO)—NR^(k)—, —S—, —SO—, —SO₂—, and —NR^(s)R^(t) (onlyif A is absent), wherein p is an integer 1 to 5; R^(k) is selected fromthe group consisting of alkyl, aryl, and hydrogen; R^(s) is selectedfrom the group consisting of hydrogen, and alkyl; R^(t) is alkyl,optionally substituted with oxo, hydroxy, methoxy, benzyloxy, halo,aryl, or heteroaryl; A is absent, or is selected from the groupconsisting of a four- to seven-membered heterocyclic ring containing 1or 2 heteroatoms independently selected from the group consisting of N,O, and S; R¹ or R⁵ is selected from the group consisting of —H, —NH₂,—(CO)—NH₂, —(CO)—NH—OH, —(CO)—NH—NH₂, —(CO)—NH—NH—R^(f), —(CO)—OH,—(CO)—O—CH₃, —(CO)—O—CH₂—CH₃, —(CO)-(4-methoxy)phenyl,—(CO)-(4-hydroxy)phenyl, —(CO)-(3-chlorophenyl), —(CO)-phenyl,—(CO)-benzyl, —(CO)—C₁₋₄alkyleneOR^(h), —(CO)—C₁₋₄alkyleneSR^(h),

—NO₂, —OH, —(CO)—C₁₋₄ alkyl, -cycloalkyl, —(CO)-substituted alkyl,—(CO)-(methoxy)alkyl, —(CO)-(alkoxy) substituted alkyl, —(CO)-aryl,—(CO)-heteroaryl, —(CO)-(substituted alkyl)_(p)-aryl, —(CO)-(substitutedalkoxy)_(p)-aryl, —(CO)-((NR^(k))_(p)-substituted alkoxy)-aryl,—(CO)-aryl-R^(d), —(CO)-aryl-R^(e), —(CO)-aryl-R^(f), —CH═N—OH,—CH═N—NH₂, —CH═N—NH—CH₃, —CH═N—NH—CH₂-phenyl, —CF₃, —(CO)—CF₃,—(CO)—CH₂-morpholinyl, —(CO)—CH₂-heteroaryl, —(CO)—CH₂—CH—(CH₃)₂,—(CO)—CH₂—CH₂—(SO₂)—CH₃, —CHO, —C≡N, —CH₂—OH, —(CO)NR^(d)R^(e),—(CS)—NH₂, —(CO)—R^(f), —(CO)—CH₂Cl, —(CO)—CH₂—NR^(d)R^(e),—(CO)—CH₂—S—(CO)—CH₃, —(CO)—CH₂—SH, —(SO₂)-phenyl,2-(anilino)-4-thiazolyl-, 2-(pyridyl)-4-thiazolyl-, -benzoxazolyl,-imidazolyl, -thiazolyl, -substituted thiazolyl, -benzimidazolyl,-benzothiazolyl, -tetrazolyl, —(N-benzyl)-tetrazolyl,—(N-methyl)-tetrazolyl, -pyrazolyl, —(N-benzyl)-pyrazolyl,—(N-methyl)-pyrazolyl, —(N-acetyl)-pyrazolyl, —(N-mesyl)-pyrazolyl,-pyrazolyl-(CO)—R^(u)R^(v), —(N-phenyl)-piperazinyl, -isoxazolyl,-pyrimidinyl, -(2-NH—CH₂-phenyl)-pyrimidinyl,-(2-(SO)-methyl)-pyrimidinyl,-(2-N-(N-t-butoxycarbonyl)-piperazinyl)-pyrimidinyl, and-(2-NH—CH₂-pyridine)-pyrimidinyl; wherein R^(d) is selected from thegroup consisting of —H, -alkyl, —CH₂-phenyl, -phenyl, —O—CH₃, -pyridyl,-thiazolyl, -thiazinyl, —O—CH₂-phenyl, —O-phenyl, —O-methoxyphenyl, —OH,—CH₂—(CO)—O—CH₃, and —CH₂—(CO)—OH; and R^(e) is selected from the groupconsisting of —H, —CH₂—CH₂—O—CH₃, —CH₂—CH₂—CH₂—N(CH₃)₂, —O—CH₃, —CH₂—CH₂(SO₂)—CH₃, O CH₃, —CH₂-pyridyl, —CH₂-phenyl, -alkyl, —CH₂—(CO)—O—CH₃,and -cylcopropyl; or R^(d) and R^(e) are taken together to form-morpholinyl, -phenylpiperazinyl, -imidazolyl, -pyrrolidinyl,—(N-methyl)-piperazinyl, and -piperidinyl; R^(f) is selected from thegroup consisting of -phenyl, -phenyl-(CF₃), -methylphenyl,-methoxyphenyl, -pyridyl, -alkyl, -benzyl, -thiophenyl, -thiazolyl,-chlorophenyl, —C(═NH)—NH₂, -fluorophenyl, —(CO)-phenyl, —(CH₂)-phenyl;R^(u) is selected from the group consisting of —H, and -alkyl; R^(v) isselected from the group consisting of —O—(CO)—CH₃, —NH-t-butoxycarbonyl,—O-phenyl, and —O—CH₂-phenyl; or R^(u) and R^(v) are taken together withthe carbon atoms to which they are attached to form a 5-membered ringcontaining an N, said N optionally protected with t-butoxycarbonyl; R²or R⁶ is selected from the group consisting of —H, —OH, -Halo, —CH₂—OH,—(CO)—NH₂, —NH₂, —(CO)—O—CH₃, —O—CH₃, —NH—(CO)—CF₃, —NH—(CO)—CH₃,—NH—(SO₂)—CH₃, —NH—CH₃, —N(CH₃)—(CO)—CF₃, —N═((CH(phenyl)-CH₂—(CO)OH,—NO₂, —O—PO₃ ^(═), —O—alkyl, —O—(CH₂)_(p)—OH, —O—(CH₂)_(p)—O-benzyl,—O—(CO)-heteroaryl, —O—(CO)-amino acid, —O—(CO)-nicotinic acid,—O—(CO)-aryl, —O—(CO)-alkyl, —O—CH₂—(CO)-benzyl, —O—(SO₂)—O—CF₃,—(CH₂)—CH═CH═N(CH₃)₂, —O—(SO₃)—, and —O—(PO)(OR^(j))(OR^(k)); whereinR^(j) independently are H, aryl, alkyl, or heterocyclic; or R¹ and R²,or R⁵ and R⁶, are taken together to form a three- or four-memberedcomponent, respectively, of a five- or six-membered ring, preferablysaid ring selected from the group consisting of -2-imidazolidonyl-,—R^(g)-thiazolyl-, -carbonylpyrrolyl methyl ketone-,-4-imino-1,3,2,-oxathiaphosphanyl-2-thione-,-4-imino-1,3,2,-oxathiaphosphanyl-2-thione-2-(4′-methoxy)phenyl-,-3-oxofuranyl-, —N-acetyl-3-oxopyrrolinyl-, —N—(CH₂—COOH)-quinolonyl-,—N-(t-butoxycarbonyl)-quinolonyl-, —N—(CH₂—COOH)-quinolinyl-,—N-(t-butoxycarbonyl)-quinolinyl-, and

wherein B is aryl or a nitrogen-containing heteroaryl, R⁹ is H orOR^(h), and R¹⁰ is selected from the group consisting of halo, OR^(h),O(CH₂)₁₋₃N(R^(h))₂, O(CH₂)₁₋₃CO₂H, CN, morpholinyl, andN-(4-methyl)-piperazinyl, wherein R^(g) is selected from the groupconsisting of -pyridyl and -anilino; R³ or R⁷, independently, isselected from the group consisting of —H, —OH, —OR^(d), —NO₂, —NH₂,—NH—R^(d), -halo, —CHO, —(SO₂)—OH, —(SO₂)—Cl, and —(SO₂)—NR^(i)R^(k);wherein R^(i) is selected from the group consisting of —H, —CH₃,—CH₂-phenyl, -phenyl, —CH₂—CH₂—O—CH₃, —CH₂—CH₂—CH₂—N(CH₃)₂, —O—CH₃,—CH₂—CH₂—(SO₂)—CH₃, -pyridyl, -thiazolyl, —O—CH₂-phenyl, —OH,—CH₂—(CO)—O—CH₃, and —CH₂—(CO)—OH; R^(k) is selected from the groupconsisting of —H, —O—CH₃, —CH₂-pyridyl, —CH₂-phenyl, —CH₃,—CH₂—(CO)—O—CH₃, -cyclopropyl, and —CH₂-cyclopropyl; or R^(j) and R^(k)are taken together to form morpholinyl, phenylpiperazinyl, imidazolyl,pyrrolidinyl, (N-methyl)-piperazinyl, and piperidinyl; and R⁴ or R⁸,independently, is selected from the group consisting of —H, —CH₃, —OCH₃,—OH, —(CO)—CH₃, -methoxyphenyl, and -pyridinyl.
 16. The pharmaceuticalcomposition of claim 12 wherein R¹ or R⁵ is selected from the groupconsisting of —H, —OH, —NH₂, —CH₂OH, —C≡N, —(CO)—NH₂, —(CO)—OH,—(CO)—O—CH₃, —CH═N—OH, —CH═N—NH₂, —CH═N—NH—CH₃, —(CO)—CF₃,—(CO)H, —NO₂,—(CO)-alkyl, —(CO)-substituted alkyl, —(CO)-aryl, —(CO)-substitutedaryl, —(CO)-heteroaryl, —(CO)—CH₂—NR^(d)R^(e), and —(CO)NR^(d)R^(e). 17.The pharmaceutical composition of claim 12 wherein R² or R⁶ is selectedfrom the group consisting of —H, —OH, —F, —CH₂—OH, —NH₂, —NH—(CO)—CF₃,—NH—(CO)—CH₃, —NH—(SO₂)—CH₃, —NH—CH₃, and —N(CH₃)—(CO)—CF₃.
 18. Apharmaceutical composition comprising: (a) a DNA-PK inhibitor of claim10, and (b) an anti-neoplastic agent.
 19. The pharmaceutical compositionof claim 18 wherein the anti-neoplastic agent comprises achemotherapeutic agent or a radiotherapeutic agent.
 20. Thepharmaceutical composition of claim 19 wherein the anti-neoplastic agentis selected from the group consisting of an alkylating agent, anantimetabolite, a type I topoisomerase inhibitor, an antimitotic drug,an antibiotic, an enzyme, a biological response modifier, adifferentiation agent, and a radiosensitizer.
 21. The pharmaceuticalcomposition of claim 19 wherein the anti-neoplastic agent is selectedfrom the group consisting of mechlorethamine, cyclophosphamide,ifosfamide, melphalan, carmustine, chlorambucil, lomustine, semustine,thriethylenemelamine, triethylene thiophosphoramide, hexamethylmelamine,busulfan, dacarbazine, methotrexate, trimetrexate, 5-fluorouracil,fluorodeoxyuridine, gemcitabine, cytosine arabinoside, 5-azacytidine,2,2′-difluorodeoxycytidine, 6-mercaptopurine, 6-thioguanine,azathioprine, 2′-deoxycofonnycin, erythrohydroxynonyladenine,fludarabine phosphate, 2-chlorodeoxyadenosine, camptothecin, topotecan,irinotecan, paclitaxel, vinblastine, vincristine, vinorelbine, docetaxeletoposide, teniposide, actinomycin D, daunomycin, doxorubicin,mitoxantrone, idarubicin, bleomycin, plicamycin, mitomycin C,dactinomycin, L-asparaginase, interferon-alpha, IL-2, G-CSF, GM-CSF,metronidazole, misonidazole, desmethylmisonidazole, pimonidazoleetanidazole, nimorazole, RSU 1069, EO9, RB 6145, SR4233, nicotinamide,5-bromodeozyuridine, 5-iododeoxyuridine, bromodeoxycytidine, cisplatin,carboplatin, mitoxantrone, hydroxyurea, N methylhydrazine, procarbazine,mitotane, aminoglutethimide, prednisone, dexamethasone,hydroxyprogesterone caproate, hydroxyprogesterone acetate, megestrolacetate, diethylstilbestrol ethynyl estradiol, tamoxifen, testosteronepropionate, fluoxymesterone, flutamide, leuprolide, flutamide, tinetioporphyrin, pheoboride-a, bacteriochlorophyll-a, a naphthalocyanine,a phthalocyanine, and a zinc phthalocyanine.
 22. A method of inhibitingDNA-PK activity comprising the step of contacting a DNA-PK with a DNA-PKinhibitor of claim 1 or
 6. 23. A method of sensitizing a cell type to anagent that induces DNA lesions comprising the step of contacting thecell type with a compound of claim 1 or
 6. 24. The method of claim 23wherein the agent that induces DNA lesions is selected from the groupconsisting of radiation, an exogenous chemical, a metabolite by-product,and combinations thereof.
 25. A method of potentiating a therapeuticregimen for treatment of a cancer comprising the step of administeringto an individual in need thereof an effective amount of a DNA-PKinhibitor of claim 1 or
 6. 26. The method of claim 25 wherein thetherapeutic regimen for treatment of cancer is selected from the groupconsisting of chemotherapy, radiation therapy, and a combination ofchemotherapy and radiation therapy.
 27. A method of characterizing thepotency of a test compound as an inhibitor of a DNA-PK polypeptide, saidmethod comprising the steps of: a) measuring an activity of a DNA-PKpolypeptide in the presence of a test compound; b) comparing theactivity of the DNA-PK polypeptide in the presence of the test compoundto the activity of the DNA-PK polypeptide in the presence of anequivalent amount of a reference compound of claim 1 or 6, wherein alower activity of the DNA-PK polypeptide in the presence of the testcompound than in the presence of the reference compound indicates thatthe test compound is a more potent inhibitor than the referencecompound, and a higher activity of the DNA-PK polypeptide in thepresence of the test compound than in the presence of the referencecompound indicates that the test compound is a less potent inhibitorthan the reference compound.
 28. A method of characterizing the potencyof a test compound as an inhibitor of a DNA-PK polypeptide, said methodcomprising the steps of: a) determining an amount of a control compoundof claim 1 or 6 that inhibits an activity of a DNA-PK polypeptide by areference percentage of inhibition, thereby defining a referenceinhibitory amount for the control compound; b) determining an amount ofa test compound that inhibits an activity of a DNA-PK polypeptide by areference percentage of inhibition, thereby defining a referenceinhibitory amount for the test compound; c) comparing the referenceinhibitory amount for the test compound to a reference inhibitory amountdetermined according to step (a) for the control compound, wherein alower reference inhibitory amount for the test compound than for thecontrol compound indicates that the test compound is a more potentinhibitor than the control compound, and a higher reference inhibitoryamount for the test compound than for the control compound indicatesthat the test compound is a less potent inhibitor than the controlcompound.
 29. The method of claim 28 wherein the method comprisesdetermining the reference inhibitory amount of the test compound in anin vitro biochemical assay.
 30. The method of claim 29 wherein themethod comprises determining the reference inhibitory amount of the testcompound in an in vitro cell-based assay.
 31. The method of claim 28wherein the method comprises determining the reference inhibitory amountof the test compound in an in vivo assay.
 32. An article of manufacturecomprising: a) an anti-cancer compound that induces double-strand DNAbreakage in cells; and b) a package insert describing a coordinatedadministration to a patient of said anti-cancer compound and a DNA-PKinhibitor compound of claim 1 or
 6. 33. The article of manufactureaccording to claim 32 wherein said anti-cancer compound induces DNAdouble strand breaks.
 34. The article of manufacture according to claim32 wherein the anti-cancer compound is selected from the groupconsisting of bleomycin and etoposide.
 35. An article of manufacture,comprising: a) a compound selected from the group consisting of acytokine, a lymphokine, a growth factor, and a hematopoietic factor; andb) a package insert describing a coordinated administration to a patientof said compound and a DNA-PK inhibitor compound of claim 1 or
 6. 36. Anarticle of manufacture comprising: a) a pharmaceutical compositioncomprising a DNA-PK inhibitor of claim 1 or 6 in a pharmaceuticallyacceptable carrier; and b) a package insert describing a therapeutictreatment comprising administering the DNA-PK inhibitor.
 37. An articleof manufacture comprising: a) a pharmaceutical composition comprising aDNA-PK inhibitor of claim 1 or 6 in a pharmaceutically acceptablecarrier; and b) a package insert describing a therapeutic treatmentcomprising administering the DNA-PK inhibitor.